C-cbl mutations and uses thereof

ABSTRACT

The present invention relates generally to the fields of molecular biology and growth factor regulation. The invention concerns methods and compositions useful for diagnosing and treating human lung cancer associated with mutated c-CBL.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and is a Continuation of U.S.application Ser. No. 13/701,569, filed Apr. 29, 2013, which claimspriority to International Application PCT/US2011/39125, filed Jun. 3,2011, which claims benefit of U.S. Provisional Application No.61/351,501, filed Jun. 4, 2010, all of which are hereby incorporatedherein by reference in their entirety.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing submitted Oct. 31, 2016 as a text file named“21117_0019U3_Updated_Sequence_Listing.txt,” created on May 13, 2016,and having a size of 18,827 bytes is hereby incorporated by referencepursuant to 37 C.F.R. § 1.52(e)(5).

BACKGROUND

Lung cancer is the second most common cancer in both men and women(after prostate and breast cancer, respectively), accounting forapproximately 15% of all new cancers. About 80% of all lung cancers arenon-small cell lung cancers (NSCLC's), which can be further divided intodistinct groups: adenocarcinoma (bronchioalveolar carcinoma as asubset), squamous cell carcinoma, and large cell carcinoma (Jemal, A. etal., Cancer statistics, 2009 July-August; 59(4):225-49). It is estimatedthat in 2009, there will be about 219,440 new cases of lung cancer. Theprognosis for NSCLC is relatively poor and a disproportionately greaterpercentage of people die due to lung cancer than prostate, breast, andcolon cancers combined. Only 15% of NSCLC patients are expected tosurvive 5 years or more, whereas the prognosis for small cell lungcancer (SCLC) is much worse. African American males have a 40% higherchance of developing lung cancer than their Caucasian counterparts(Abidoye, O. et al. Nat Clin Pract Oncol. 2007 February; 4(2):118-29).Additionally, they also suffer from much higher morbidity and mortalityfrom this disease. Given the incidence of NSCLC and its ethnic disparityand poor prognosis, there is clearly an urgent need to identify anddevelop novel targeted therapies that are especially directed at theAfrican American patient population. Gender differences are alsostriking, with women having significantly better prognosis as comparedto men. There are a number of genetic alterations that can occur in lungcancer. As an example, in NSCLC, mutations in KRAS, p53, EGFR and METhave been identified. Many of the pathways that involve these genes andtheir corresponding proteins, especially Receptor Tyrosine Kinases(RTKs), are controlled by c-CBL.

CBL (Casitas B-lineage lymphoma) is a mammalian gene located on humanchromosome 11q23.3.), and its protein product is involved in cellsignaling and protein ubiquitination (Swaminathan, G. et al., 2006). CBLproteins belong to the RING finger class of ubiquitin ligases (E3) andthere are three homologues: c-CBL, CBL-b, CBL-3. The c-CBL and CBL-bgenes are ubiquitously expressed with the highest levels inhematopoietic tissues (Kozlov G, et al. 2007). c-CBL consists of fourregions encoding for functionally distinct protein domains: theN-terminal tyrosine kinase binding (TKB) domain, the linker region, thecatalytic RING finger domain, the proline-rich region and the c-terminalubiquitin-associated (UBA) domain that also overlaps with aleucine-zipper (LZ) domain (Swaminathan G, and Tsygankov A Y. (2006) JCell Physiol 209:21-43). Both TKB and RING finger domains are essentialfor ligand-induced ubiquitination of RTKs (Lill N L, et al. 2000). Theevolutionarily conserved N-terminal region of CBL is sufficient toenhance down-regulation of the epidermal growth factor receptor (MiyakeS, et al., J Biol Chem 275:367-377 (1999)). It is the RING finger ofc-CBL that mediates desensitization of the epidermal growth factorreceptor. RING finger mutations can abolish c-CBL-directedpolyubiquitination and downregulation of the EGF receptor, but these areinsufficient for cell transformation (Waterman H, et al., Mol Cell7:355-365; (1999)). The TKB domain includes four-helix bundle (4H), acalcium-biding EF hand, and a modified SH2 domain, which binds tophosphotyrosine residues.

The CBL family, especially c-CBL, has been recognized as a key player inthe negative regulation of antigen receptor and other signalingpathways. (Swaminathan and Tsygankov, 2006). In addition, c-CBL has beenpointed out as a regulator of RTKs (Thien C B, and Langdon W Y. 2001.Nat Rev Mol Cell Biol 2:294-307), with many studies indicating thatc-CBL plays an important role in downregulation of RTKs such as c-Metand epidermal growth factor receptor (EGFR) through its E3 ubquitinligase activity. (Pennock S. and Wang Z. 2008. Mol Cell Biol.28(9):3020-37).

c-Met is a RTK, which stimulates the invasive growth of carcinoma cells,and is tumorigenetically mutated and overexpressed in many solid tumors.Overexpression of c-Met has been shown in small cell lung cancer (SCLC)and non-small cell lung cancer (NSCLC) cells. In addition, studies haveidentified C-Met mutations located in specific domain, e.g. thejuxtamembrane domain, which results in the loss of c-CBL E3-ligasebinding.

EGFR, a member of the ErbB family, is important in the regulation ofcell growth, differentiation and survival. EGFR overexpression isdetected in various types of malignant tumors; thus, EGFR is a promisingtherapeutic target. Mutations in the EGFR kinase domain are associatedwith clinical response to EGFR inhibitors and are observed frequently inNSCLC patients in East Asian populations.

As is the case with other cancers, lung cancers are also addicted to avariety of oncogenes and growth factors. EGFR is one of the best-studiedRTKs in the context of NSCLC. EGFR frequently becomes overexpressed inNSCLC and can acquire gain-of-function mutations, and importantly bothof these events can occur concurrently. The above modifications confergrowth and invasive advantages to lung tumors. Another RTK that isoverexpressed and frequently acquires gain-of-function mutations isc-MET. More than EGFR, c-MET promotes cell motility and migration,thereby contributing significantly to metastasis. In addition to thec-MET receptor, its natural ligand hepatocyte growth factor (HGF) isalso frequently overexpressed (10 to 100 fold higher) in lung cancerscompared to adjacent normal tissue. Higher levels of HGF are associatedwith more aggressive tumor biology and a poorer prognosis in NSCLC. TheHGF/c-MET autocrine loop plays a vital role in the epithelialmesenchymal transition (EMT) that underlies the metastasis process.

In a normal cell, the steady state level of any RTK is the net balancebetween synthesis and degradation. The synthesis of EGFR and c-MET inNSCLC is generally boosted by aberrant gene amplification. Although thefrequent mutations seen in the kinase domains of EGFR and c-MET areknown to contribute to increased tyrosine kinase activity, mutations inthe juxtamembrane region, especially in c-MET, result in loss ofnegative regulation. The functionality of RTKs is known to bedownregulated in normal cells through ubiquitination-mediatedproteasomal degradation. Most of the RTKs such as EGFR, c-MET, KIT andIGFR are negatively regulated by a specific E3 ubiquitin ligase: c-CBL.

c-CBL also binds to EGFR and acts as the E3 that targets EGFR forubiquitination and degradation, thereby desensitizing EGF signaling andopposing induced by EGF-induced cellular proliferation. EGF activationalso appears to activate the tyrosine kinase SRC, which phosphorylatesc-CBL and in turn activates the ubiquitination and degradation of EGFR.Specifically, initial binding of c-CBL to EGFR is either by directassociation between the tyrosine phosphorylated receptor and c-CBL TKdomain, or it is facilitated by the adaptor Grb2. The SH2 domain of Grb2binds to the phosphorylated receptor and the SH3 region interacts withproline-rich region of c-CBL. EGFR mediated tyrosine phosphorylation ofc-CBL results in activation of the ligase and mediatesmulti-monoubiquitylation of the K48 residues. This triggers the‘destruction sequence’ starting with endocytosis of EGFR throughclathrin-coated pits. CIN85 provides the link between the c-CBL/EGFRcomplex and the endocytic protein endophilin. The trafficking of thereceptor to the lysosome culminates in its degradation. In contrast,non-ubiquitinated receptors are effectively internalized, and recycledback to the plasma membrane. A similar ‘destruction sequence’ andrecycling process have been reported for MET. In short, activation ofEGFR or MET by ligand binding under physiological conditions results inrecruitment of the c-CBL ubiquitin ligase in conjunction with an E2ubiquitin-conjugating enzyme. More detailed studies have demonstratedthat c-CBL mediates monoubiquitinaton or polyubiquitination of receptorsand initiates receptor endocytosis. Receptor bound c-CBL also recruitsCIN-85/endophilin-A1 complexes to the plasma membrane which is necessaryfor invagination and formation of endocytic vesicles.

A recent study shows that defective endocytosis of EGFR is characterizedby a deletion mutant and the point mutation L858R, whereby itsassociation with c-CBL and subsequent ubiquitination are impaired.Recently, the first human c-CBL mutations were reported in acute myeloidleukemia (AML) patients.

Not only can E3 activity be important in oncogenesis, c-CBL has a dualbut separate function as a signal transduction molecule. c-CBL haspreviously been shown to be important in binding CRKL and BCR/ABL inhematopoietic cells. Also, it can bind and modulate functions ofcytoskeleton by binding to proteins like talin and paxillin. The TKBdomain is important in binding to a number of molecules that function insignal transduction.

As described above, c-Met also plays a role in the development andprogression of cancer and represents a therapeutic target. Unlikeimatinib for CML (targeting Bcr/Abl) and gastrointestinal stromal tumors(GIST; targeting c-Kit), targeted small molecule inhibitors againstc-Met have yet to be approved for use in humans. Several c-Metinhibitors are currently in clinical development. Also an antagonist ofHGF, NK4, was previously reported to be generated by proteolyticdigestion of HGF (Date, et al., FEBS Lett, 420, (1), 1-6 (1997)). NK4 isa truncated HGF composed of the NH2-terminal hairpin domain and fourkringle domains in the alpha-chain of HGF. It retains c-Met receptorbinding properties without mediating biological responses. NK4antagonizes HGF-induced tyrosine phosphorylation of c-Met, resulting ininhibition of HGF-induced motility, angiogenesis and invasion of HT115human colorectal cancer cells (Parr, et al., Int J Cancer, 85, (4),563-70 (2000)). Also, when administered to pancreatic tumor-bearingmice, NK4 inhibited growth, invasion, and disseminating metastasis ofpancreatic cancer cells, and prolonged the lifespan of these mice(Tomioka, et al., Cancer Res, 61, (20), 7518-24 (2001)). Finally, asoluble chimeric form of c-Met was shown to retain full capacity to bindHGF and therefore neutralize HGF activity Mark, et al., J Biol Chem,267, (36), 26166-71 (1992)). NK4, pro-HGF (uncleavable HGF) and thedecoy c-Met receptor have been shown to inhibit mutant c-Met-inducedtransformation of NIH3T3 cells (Michieli, et al., Oncogene, 18, (37),5221-31 (1999)).

Small molecule inhibitors directed specifically against c-Met representan attractive therapeutic approach. The effectiveness of a novelspecific small molecule inhibitor of c-Met, SU11274 was first reportedby Sattler, et al. (Pfizer; previously Sugen), in cells transformed bythe oncogenic Tpr-Met as a model, as well as in SCLC (Sattler, et al.,Cancer Res, 63, (17), 5462-9 (2003)). Inhibition of the Met kinaseactivity by the drug SU11274 led to time- and dose-dependent reducedcell growth and induced G1 cell cycle arrest and apoptosis (Ma, et al.,Cancer Res, 65, (4), 1479-88 (2005)). Met kinase autophosphorylation wasreduced on sites that have been previously shown to be important foractivation of pathways involved in cell growth and survival, especiallythe phosphatidylinositol-3′-kinase (PI3K) and the Ras pathway. Thecharacterization of SU11274 as an effective inhibitor of Met tyrosinekinase activity illustrates the therapeutic potential of targeting Metin cancers associated with activated forms of this kinase.

To impact on this disease, newer and novel targeted therapies need to beemployed. However, it still remains to be seen how and if patientsrespond to such inhibitors.

Even with the best therapies and recent advent of novel molecularlytargeted therapies, overall survival for all Non-Small Cell Lung Cancer(NSCLC) patients is only 15% over a five year period. Receptor tyrosinekinases (RTKs) have shown to be important in a variety of malignancies,such as c-Kit in GISTs and epidermal growth factor receptor (EGFR) inNSCLC. However, the response to EGFR blockade by small moleculeinhibitors, such as erlotinib, is at best 5-15% in refractory advancedNSCLC. The compositions and methods disclosed herein will provide ameans to address such issues.

Head and neck squamous cell carcinoma (HNSCC) is a heterogenous group ofdisorders in which RTKs such as EGFR and MET are overexpressed. c-CBL isinvolved in the degradation of receptor tyrosine kinases via targetingfor lysosomal-mediated degradation in HNSCC. c-CBL protein expression islargely reduced or absent in HNSCC patient tumor specimens, and this wascorrelated with increased expression of MET. This pattern of c-CBL andMET protein expression is largely recapitulated in HNSCC cell lines. InHNSCC tumor specimens, c-CBL was found to be mutated, and LOH wasdetected at the c-CBL locus. Additionally, MET is activated in HNSCC,and pMET expression is concomitant with MET expression. These datasupport the notion that diminished c-CBL expression in HNSCC is relatedto the increased expression of MET. In certain hematologic malignanciesc-CBL has Uniparental Disomy with activating c-CBL mutations. Therelative low expression level of c-CBL implicates this molecule as atumor suppressor in HNSCC; whereas in other tumors it likely functionsas an adaptor molecule, in which case it has been shown previously thatBCR/ABL utilizes c-CBL for a plethora of signal transduction. In cellculture models of HNSCC in which c-CBL was knocked down using targetedsiRNA, MET expression was increased and cell viability was decreased.MET is largely overexpressed in HNSCC, and it can be effectivelytargeted using small molecule chemical inhibitors.

All references cited herein, including patent applications andpublications, are incorporated by reference in their entirety.

Additional advantages of the invention will be set forth in part in thedescription which follows, and in part will be obvious from thedescription, or may be learned by practice of the invention. Theadvantages of the invention will be realized and attained by means ofthe elements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention, as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments of theinvention and together with the description, serve to explain theprinciples of the invention. These are non-limiting examples.

FIGS. 1A and 1B show c-CBL mutations. FIG. 1A shows chromatogramsindicating the location of two exon mutations that were analyzed asdescribed in Example 1. The chromatograms showed the patient withsomatic mutation Q249E had nucleotide C>CA changes and the patient withSNP L620F had nucleotide C>CT changes. The arrows indicate the changednucleotide sites. FIG. 1B shows the c-CBL domain structure that wasanalyzed during the experimental example of FIG. 1A. As depicted in FIG.1B, c-CBL has N-terminal tyrosine kinase binding (TKB) domain, RINGfinger domain, proline rich (Pro_rich) region, and C-terminalubiquitin-associated (UBA) domain. The mutations Q249E and L620F arelocated on TKB domain and Pro_rich region, respectively.

FIG. 2 shows the c-CBL mutation rates for the different tumor types thatwere studied in as described in Example 1. The mutation rates foradenocarcinoma (AD), squamous cell carcinoma (SQ), adenosquamouscarcinoma (AD/SQ), and large cell carcinoma (LC) patients were 77.8%,100%, 33.3%, and 66.7%, respectively. The statistical analysis showedthat the mutation rates between AD and SQ (P=0.133), between AD andAD/SQ (P=0.058), and between AD and LC (P=0.599) were not significant.

FIG. 3 shows c-CBL wild-type and mutant constructs that were made into apcDNA3.1-NmCherry vector during the experimental example of FIGS. 1 and2. The pcDNA3.1-NmCherry vector had ampicillin and neomycin forantibiotic selection. The multiple cloning site for pcDNA3.1(+)contained mCherry inserted into the ApaI site, and a cDNA sequence ofc-CBL wild-type and Q249E mutant was inserted between BamHI and XhoI.The vector map was modified from http://www.invitrogen.com.

FIG. 4 shows the A549 cell viability of c-CBL wild-type and Q249E mutantconstructs that were studied during the experimental example of FIGS.1-3. Compared to the control empty vector (EV) transfectant, c-CBLwild-type (WT) and mutant Q249E transfectants showed 102.5% (P=0.868)and 156% (P=0.049) cell viability in A549 cells after 48 h transfection.

FIGS. 5A, 5B, 5C, and 5D show c-CBL mutants. FIG. 5A is a schematicillustration of various c-CBL mutants with respect to the differentfunctional domains that were analyzed as described in Example 2. Threemutations: S80N, H94Y, Q249E were located on the TKB domain; V391I waslocated on the RING finger domain; 72515_72517 del ATG (a non-frameshiftdeletion) and L620F (a known SNP, rs2227988) were located on Pro-richregion, and W802*, R830K, and A848T were found in the C-terminal regionof c-CBL. The numbers shown represent amino acid positions. Associationof c-CBL mutations among different ethnic populations is shown in thetable in FIG. 5A. FIG. 5B depicts representative examples of sequencingchromatograms of the mutation region in normal (N) and tumor (T) samplesthat were analyzed in the experimental example of FIG. 5A. Arrowsindicate the heterozygous mutation in the tumor sample. FIG. 5Cgraphically summarizes a Loss of Heterozygosity (LOH) analysis of 37tumor and paired normal samples from Taiwanese patients that wereanalyzed in the experimental example of FIGS. 5A and 5B. A value of <0.5indicates a LOH at the c-CBL locus. FIG. 5D is a schematic depiction ofChromosome 11 markers chosen for LOH analysis in the experimentalexample of FIGS. 5A-5C, as well as representative examples of LOHchromatogram analysis. After amplification using chromosome 11-specificmicrosatellite primers, the PCR product was separated by capillaryelectrophoresis, and bands were quantified according to the intensity.

FIG. 6 shows the relationship between c-CBL mutations and MET and EGFRmutations in lung cancer, as analyzed as described in Example 2. 37Taiwanese samples were analyzed for mutations in c-CBL, MET and EGFR andfor LOH analysis in the c-CBL locus. Data shows 21.6% (8/37) had LOH,whereas 8% (3/37) had a c-CBL mutation (including the known SNP L620F),these samples were mutually exclusive. Additionally, 5 of 8 LOH samplesalso had MET N375S mutation and 2 had an EGFR exon 19 deletion. In thesamples having c-CBL mutation, 1 also had a MET N375S mutation whileanother had an EGFR L858R mutation. In samples that did not harbor anyc-CBL mutation (70%, 26/37), 22 had either a MET or an EGFR mutation andonly 4 did not have a mutation in any of these 3 genes.

FIGS. 7A, 7B, 7C, and 7D show cellular data on four c-CBL mutants. FIG.7A shows western blots of whole cell extracts from A549 cells that hadbeen transfected with one of four c-CBL genes (wild-type (WT), Q249E,W802Stop, or S80N/H94Y) in the context of the pAlterMax vector, asdescribed in Example 2 below. FIG. 7A, top, shows that c-CBL mutants didnot alter ubiquitination of EGFR. Cells were co-transfected with EGFR,and different c-CBL mutants were stimulated with EGF, immunoprecipitatedwith anti-EGFR antibody, and blotted with anti-ubiquitin antibody.Immunoblot with anti-EGFR antibody served as the IP control, while theanti-HA blot was used as the input control. FIG. 7B shows the cellviability measurements for these clones. Cell viability was measured byTrypan blue exclusion and compared to empty vector control. c-CBLwild-type (WT) and mutants S80N/H94Y, Q249E, and W802* showed 66.7%,132.3%, 120.8%, and 147.9% cell viability, respectively, in A549 cells48 h after transfection. Experiments were performed in triplicate, andthe mean data is shown. Error bars indicate the Standard Deviation. FIG.7C displays c-CBL and beta-actin protein expression levels of thevarious mutants that were analyzed by Western blots using theappropriate antibodies. FIG. 7D graphically displays cell cycle analysisof the different c-CBL mutants 48 h after transfection in A549 cells.

FIGS. 8A and 8B provide wound healing data. FIG. 8A shows the results ofa wound healing assay that was performed in A549 cells that had beentransfected with one of four c-CBL genes (wild-type (WT), Q249E, W802*,or S80N/H94Y) in the context of the pAlterMax vector, as described inExample 2 below. Empty vector (EV) was used as a control. Representativepictures (Brightfield and phase contrast) from each time point areshown. FIG. 8B displays a graph of the open wound percentage at eachtime point. The open wound at each time point was quantified andnormalized to 0 h. Experiments were done in triplicate, and the meandata is shown. Error bars indicate Standard Deviation.

FIGS. 9A and 9B show data using H358 cell line clones. FIG. 9A showsimages of Western blots of several H358 cell line clones showed that thec-CBL lentiviral shRNA knockdown efficiency varied among the variousclones (FIG. 9A), as described in Example 2 below. Scrambled shRNA (Scr)was used as a control. Of all the clones tested, Clone 27, whichpresented the highest knockdown efficiency, was chosen for furtherexperiments, presented in FIG. 9B. FIG. 9B is a graph showing the cellproliferation percentage for H358 clone 27 cells transfected with shRNAdirected toward c-CBL. Lung cancer cell line H358 clone 27 stablytransfected with shRNA showed an increase in cell counts compared to thescrambled shRNA control. Experiments were done in triplicates, and themean data is shown. Error bars indicate Standard Deviation.

FIGS. 10A and 10B show MET and c-CBL staining. FIG. 10A shows images ofMET and c-CBL staining in an adenocarcinoma sample and in a NSCLC sampleanalyzed as described in Example 3 below. Adenocarcinoma andundifferentiated NSCLC were stained with c-CBL and MET antibodies onwhole tissue sections. c-CBL staining was diffuse but weak, whereas METstaining in the tumor was strong (T, tumor). The darker areas of c-CBLstaining were localized to the lymphocytes (L), whereas no MET stainingwas detected in lymphocytes. FIG. 10B is a bar graph showing relativestaining intensity of MET and c-CBL in tumor sections of samples (n=29,3=intense, 2=moderate, 1=weak). MET staining was more intense insquamous cell carcinoma (SQ, n=11) and adenocarcinoma (AD, n=11)compared to c-CBL.

FIG. 11 shows CBL isoform staining in the lung cancer cells that wereanalyzed as described in Example 3, below. The bar graph shows theintensity of c-CBL (n=12) staining compared to CBL-b (n=12) and CBL-c(n=12) staining in lung cancers (Adapted from www.proteinatlas.org). Asdepicted, most lung cancer tissues had no or low c-CBL staining but highCBL-b and CBL-c staining.

FIG. 12 shows images of a tumor growth and xenograft model that wasanalyzed in Example 4. Tumor growth of luc gene stably transfected intoNSCLC (A549 luc) xenograft in ectopic (left) and metastatic (right)athymic nude mice models. Tumor and metastatic A549 luc tumor noduleswere visible on the lung surfaces (shown in the lower panel).

FIG. 13 shows that knockdown of c-CBL in H358 cells reduced the cells'susceptibility to killing by a c-MET inhibitor, as described in Example5, herein. H358 clone 27 cells expressing high levels of c-CBL wereknocked down using a lentiviral construct (sh 27). Cells were treatedwith the indicated RTK inhibitors, PI-3K inhibitors or cisplatin (CDDP).Live cells were assessed 72 h after drug treatment using a MTT assay.

FIG. 14 shows the positions of five c-CBL mutations found in head andneck cancer, four in the TKB region (P170L, S171S, L281F, L254S) and onein the C-terminal region (P782L). The mutations were identified inpatient samples. All mutations were in different patients.

FIGS. 15A, 15B, and 15C show information related to the expression ofc-CBL and MET in head and neck squamous cell carcinomas (HNSCC) tumorspecimens. FIG. 15A shows representative IHC images of c-CBL, MET, andp-MET expression in HNSCC tumor specimens. FIGS. 15B and C shows pooledtissue microarray (TMA) information related to the expression of c-CBL,c-Met and phosph c-Met. This Figure shows the staining intensity scoreas described in Example 4 versus the percentage of samples for CBL,c-met and p-met.

FIG. 16 shows CBL versus MET expression in each sample. The average Metand CBL TMA staining intensity scores in the same patient sample forseveral samples.

FIG. 17 shows information related to c-CBL and MET expression in HNSCCcell lines. Whole cell lysates from 11 HNSCC cell lines were subjectedto SDS-PAGE, then immunoblotted using the indicated antibodies. β-actinserved as the loading control.

FIGS. 18A and 18B show c-CBL mutations in HNSCC. FIG. 18A shows aschematic of the functional domains of the c-CBL protein and thelocation of the mutations identified in 2/20 HNSCC tumor specimens. FIG.18B shows representative sequencing chromatograms of the mutation regionin normal (N) and tumor (T) samples.

FIGS. 19A and 19B show information related to LOH at the c-CBL locus inHNSCC. LOH analysis of 23 tumor and paired normal patient samples. AfterPCR amplification using chromosome 11 specific microsatellite primers,the PCR product was separated by capillary electrophoresis and bandswere quantified according to intensity. FIG. 19A shows a schematic ofchromosome 11 with location of primers and representative examples ofLOH chromatogram analysis. FIG. 19B shows a summary bar graph of LOHresults. A ratio of Tumor:Normal <0.5 indicates LOH at the c-CBL locus.

FIG. 20 shows the effect of representative c-CBL mutants, S80N/H94Y (SH)double mutation, Q249E (Q), V391I (V) and W802* (W*), on lung cancercell viability in A549 and H226 nonsmall cell lung cancer cell lines.

FIG. 21 shows immunoprecipitations (IP) with anti-c-Met Ab andimmunoblots (IB) with anti-ubiquitin Ab of A549 cells transientlytransfected with empty vector (EV) or c-CBL wild type (WT) and mutants'constructs (SH: S80N/H94Y, Q: Q249E, V: V391I, W*: W802*). The resultsshowed the ubiquitination of c-Met were decreased in A549 cells.

FIG. 22 shows the sensitivity of cancer cells to specific cancertherapeutics. The results in A549 cells showed c-CBL mutants had moresensitivity to c-Met inhibitor SU11274 than wild type c-CBL. The EGFRinhibitor, Tarceva, showed no differential effects on the A549 wild typeand mutant c-CBL-transfected cells.

FIG. 23 shows the sensitivity of A549 cells containing of representativec-CBL mutants, S80N/H94Y (SH) double mutation, Q249E (Q), V391I (V) andW802* (W*) to 1 μM SU11274. c-CBL mutants are more sensitive than wildtype c-CBL.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure describes, at least in part, the discovery ofmultiple mutational events in the E3 ubiquitin ligase, c-CBL. It waspreviously thought that aberrant c-Met activity was associated withvarious cancers, and c-Met became a potential therapeutic target fortreating or preventing cancer; however, it was unknown what, if any,treatments would be effective for any given subject.

Disclosed herein are methods of identifying a subject that issusceptible to treatment with a c-Met inhibitor. For example, disclosedherein are methods of identifying a subject that is susceptible totreatment with a c-Met inhibitor comprising determining whether a samplefrom the subject comprises a mutation in a nucleic acid sequenceencoding human c-CBL, wherein the mutation results in an amino acidchange at position S80N, H94Y, Q249E, V391I, 72515_72517delATG, W802*,R830K, A848T, L620F, P170L, S171S, L281F, L254S, or P782L.

Also disclosed herein are methods of identifying a subject that issusceptible to treatment with a c-Met inhibitor comprising determiningwhether a sample from the subject comprises a mutation in a nucleic acidsequence encoding human c-CBL, wherein the mutation is located in theTKB domain, RING finger domain, proline-rich region, C-terminal region,or other domain linkage regions of c-CBL.

Also disclosed herein are methods of identifying a cancer that issusceptible to treatment with a c-Met inhibitor, comprising determiningwhether a sample from the cancer comprises a mutation in a nucleic acidsequence encoding human c-CBL.

Also disclosed herein are methods of determining responsiveness of acancer in a subject to treatment with a c-Met inhibitor, said methodcomprising determining whether a cancer sample from a subject comprisesa mutation in a nucleic acid sequence encoding human c-CBL, wherein themutation results in an amino acid change at position S80N, H94Y, Q249E,V391I, 72515_72517delATG, W802*, R830K, A848T, L620F, P170L, S171S,L281F, L254S, or P782L, wherein the presence of the mutated nucleic acidsequence is indicative that the cancer is responsive to treatment withthe c-Met inhibitor.

Also disclosed herein are methods of determining responsiveness of acancer in a subject to treatment with a c-Met inhibitor, said methodcomprising determining whether a cancer sample from a subject comprisesa mutation in a nucleic acid sequence encoding human c-CBL, wherein themutation is located in the TKB domain, RING finger domain, proline-richregion, C-terminal region, or other domain linkage regionsv of c-CBL,wherein the presence of the mutated nucleic acid sequence is indicativethat the cancer is responsive to treatment with the c-Met inhibitor.

Also disclosed herein are methods of detecting cancer in a samplecomprising determining whether the sample comprises a mutation in anucleic acid sequence encoding human c-CBL, wherein the mutation resultsin an amino acid change at position S80N, H94Y, Q249E, V391I,72515_72517delATG, W802*, R830K, A848T, L620F, P170L, S171S, L281F,L254S, or P782L.

Also disclosed herein are methods of detecting cancer in a samplecomprising determining whether the sample comprises a mutation in anucleic acid sequence encoding human c-CBL, wherein the mutation islocated in the TKB domain, RING finger domain, proline-rich region,C-terminal region, or other domain linkage regions of the nucleic acidsequence encoding human c-CBL.

All patents, patent applications and publications cited herein, whethersupra or infra, are hereby incorporated by reference in their entiretiesinto this application in order to more fully describe the state of theart as known to those skilled therein as of the date of the inventiondescribed and claimed herein.

It is to be understood that this invention is not limited to specificsynthetic methods, or to specific recombinant biotechnology methodsunless otherwise specified, or to particular reagents unless otherwisespecified, to specific pharmaceutical carriers, or to particularpharmaceutical formulations or administration regimens, as such may, ofcourse, vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only andis not intended to be limiting.

Definitions and Nomenclature

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting.

The invention relates to the discovery that mutations in c-CBL play arole in cancer and have an effect on the effect of c-Met inhibitors.c-CBL refers to the polypeptide encoded by a c-CBL gene. Both of theseterms are used herein as general identifiers. Thus, for example, a c-CBLgene or nucleic acid refers to any gene or nucleic acid identified withor derived from a wild-type or mutated c-CBL gene. For example, a mutantc-CBL gene is a form of c-CBL gene.

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” can include plural referents unless the contextclearly dictates otherwise. Thus, for example, reference to “a compound”includes mixtures of compounds; reference to “a pharmaceutical carrier”includes mixtures of two or more such carriers, and the like.

Ranges may be expressed herein as from “about” one particular value,and/or to “about” another particular value. The term “about” is usedherein to mean approximately, in the region of, roughly, or around. Whenthe term “about” is used in conjunction with a numerical range, itmodifies that range by extending the boundaries above and below thenumerical values set forth. In general, the term “about” is used hereinto modify a numerical value above and below the stated value by avariance of 20%. When such a range is expressed, another embodimentincludes from the one particular value and/or to the other particularvalue. Similarly, when values are expressed as approximations, by use ofthe antecedent “about,” it will be understood that the particular valueforms another embodiment. It will be further understood that theendpoints of each of the ranges are significant both in relation to theother endpoint, and independently of the other endpoint.

The amino acid abbreviations used herein are conventional one lettercodes for the amino acids and are expressed as follows: A, alanine; B,asparagine or aspartic acid; C, cysteine; D aspartic acid; E, glutamate,glutamic acid; F, phenylalanine; G, glycine; H histidine; I isoleucine;K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q,glutamine; R, arginine; S, serine; T, threonine; V, valine; W,tryptophan; Y, tyrosine; Z, glutamine or glutamic acid.

“Polypeptide” as used herein refers to any peptide, oligopeptide,polypeptide, gene product, expression product, or protein. A polypeptideis comprised of consecutive amino acids. The term “polypeptide”encompasses naturally occurring or synthetic molecules.

In addition, as used herein, the term “polypeptide” refers to aminoacids joined to each other by peptide bonds or modified peptide bonds,e.g., peptide isosteres, etc. and may contain modified amino acids otherthan the 20 gene-encoded amino acids. The polypeptides can be modifiedby either natural processes, such as post-translational processing, orby chemical modification techniques which are well known in the art.Modifications can occur anywhere in the polypeptide, including thepeptide backbone, the amino acid side-chains and the amino or carboxyltermini. The same type of modification can be present in the same orvarying degrees at several sites in a given polypeptide. Also, a givenpolypeptide can have many types of modifications. Modifications include,without limitation, acetylation, acylation, ADP-ribosylation, amidation,covalent cross-linking or cyclization, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of a phosphytidylinositol,disulfide bond formation, demethylation, formation of cysteine orpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristolyation, oxidation, pergylation, proteolytic processing,phosphorylation, prenylation, racemization, selenoylation, sulfation,and transfer-RNA mediated addition of amino acids to protein such asarginylation. (See Proteins—Structure and Molecular Properties 2nd Ed.,T. E. Creighton, W.H. Freeman and Company, New York (1993);Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York, pp. 1-12 (1983)).

As used herein, the term “amino acid sequence” refers to a list ofabbreviations, letters, characters or words representing amino acidresidues.

As used herein, “peptidomimetic” means a mimetic of a peptide whichincludes some alteration of the normal peptide chemistry.Peptidomimetics typically enhance some property of the original peptide,such as increase stability, increased efficacy, enhanced delivery,increased half life, etc. Methods of making peptidomimetics based upon aknown polypeptide sequence are described, for example, in U.S. Pat. Nos.5,631,280; 5,612,895; and 5,579,250. Use of peptidomimetics can involvethe incorporation of a non-amino acid residue with non-amide linkages ata given position. One embodiment of the present invention is apeptidomimetic wherein the compound has a bond, a peptide backbone or anamino acid component replaced with a suitable mimic. Some non-limitingexamples of unnatural amino acids which may be suitable amino acidmimics include β-alanine, L-α-amino butyric acid, L-γ-amino butyricacid, L-α-amino isobutyric acid, L-ϵ-amino caproic acid, 7-aminoheptanoic acid, L-aspartic acid, L-glutamic acid,N-ϵ-Boc-N-α-CBZ-L-lysine, N-ϵ-Boc-N-α-Fmoc-L-lysine, L-methioninesulfone, L-norleucine, L-norvaline, N-α-Boc-N-δCBZ-L-ornithine,N-δ-Boc-N-α-CBZ-L-ornithine, Boc-p-nitro-L-phenylalanine,Boc-hydroxyproline, and Boc-L-thioproline.

The word “or” as used herein means any one member of a particular listand also includes any combination of members of that list.

The phrase “nucleic acid” as used herein refers to a naturally occurringor synthetic oligonucleotide or polynucleotide, whether DNA or RNA orDNA-RNA hybrid, single-stranded or double-stranded, sense or antisense,which is capable of hybridization to a complementary nucleic acid byWatson-Crick base-pairing. Nucleic acids of the invention can alsoinclude nucleotide analogs (e.g., BrdU), and non-phosphodiesterinternucleoside linkages (e.g., peptide nucleic acid (PNA) orthiodiester linkages). In particular, nucleic acids can include, withoutlimitation, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA or any combinationthereof

As used herein, “reverse analog” or “reverse sequence” refers to apeptide having the reverse amino acid sequence as another, reference,peptide. For example, if one peptide has the amino acid sequence ABCDE,its reverse analog or a peptide having its reverse sequence is asfollows: EDCBA.

By “sample” is meant an animal; a tissue or organ from an animal; a cell(either within a subject, taken directly from a subject, or a cellmaintained in culture or from a cultured cell line); a cell lysate (orlysate fraction) or cell extract; or a solution containing one or moremolecules derived from a cell or cellular material (e.g. a polypeptideor nucleic acid), which is assayed as described herein. A sample mayalso be any body fluid or excretion (for example, but not limited to,blood, urine, stool, saliva, tears, bile) that contains cells or cellcomponents.

By “modulate” is meant to alter, by increase or decrease.

By “normal subject” is meant an individual who does not have cancer aswell as an individual who has increased susceptibility for developing acancer.

By an “effective amount” of a compound as provided herein is meant asufficient amount of the compound to provide the desired effect. Theexact amount required will vary from subject to subject, depending onthe species, age, and general condition of the subject, the severity ofdisease (or underlying genetic defect) that is being treated, theparticular compound used, its mode of administration, and the like.Thus, it is not possible to specify an exact “effective amount.”However, an appropriate “effective amount” may be determined by one ofordinary skill in the art using only routine experimentation.

By “isolated polypeptide” or “purified polypeptide” is meant apolypeptide (or a fragment thereof) that is substantially free from thematerials with which the polypeptide is normally associated in nature.The polypeptides of the invention, or fragments thereof, can beobtained, for example, by extraction from a natural source (for example,a mammalian cell), by expression of a recombinant nucleic acid encodingthe polypeptide (for example, in a cell or in a cell-free translationsystem), or by chemically synthesizing the polypeptide. In addition,polypeptide fragments may be obtained by any of these methods, or bycleaving full length polypeptides.

By “isolated nucleic acid” or “purified nucleic acid” is meant DNA thatis free of the genes that, in the naturally-occurring genome of theorganism from which the DNA of the invention is derived, flank the gene.The term therefore includes, for example, a recombinant DNA which isincorporated into a vector, such as an autonomously replicating plasmidor virus; or incorporated into the genomic DNA of a prokaryote oreukaryote (e.g., a transgene); or which exists as a separate molecule(for example, a cDNA or a genomic or cDNA fragment produced by PCR,restriction endonuclease digestion, or chemical or in vitro synthesis).It also includes a recombinant DNA which is part of a hybrid geneencoding additional polypeptide sequence. The term “isolated nucleicacid” also refers to RNA, e.g., an mRNA molecule that is encoded by anisolated DNA molecule, or that is chemically synthesized, or that isseparated or substantially free from at least some cellular components,for example, other types of RNA molecules or polypeptide molecules.

By a “transgene” is meant a nucleic acid sequence that is inserted byartifice into a cell and becomes a part of the genome of that cell andits progeny. Such a transgene may be (but is not necessarily) partly orentirely heterologous (for example, derived from a different species) tothe cell.

By “transgenic animal” an animal comprising a transgene as describedabove. Transgenic animals are made by techniques that are well known inthe art.

By “knockout mutation” is meant an alteration in the nucleic acidsequence that reduces the biological activity of the polypeptidenormally encoded therefrom by at least 80% relative to the unmutatedgene. The mutation may, without limitation, be an insertion, deletion,frameshift, or missense mutation. A “knockout animal,” for example, aknockout mouse, is an animal containing a knockout mutation. Theknockout animal may be heterozygous or homozygous for the knockoutmutation. Such knockout animals are generated by techniques that arewell known in the art. A preferred form of knockout mutation is onewhere the biological activity of the c-CBL polypeptide is not completelyeliminated.

By “treat” is meant to administer a compound or molecule to a subject,such as a human or other mammal (for example, an animal model), that hasan increased susceptibility for developing a cancer, or that has acancer, in order to prevent or delay a worsening of the effects of thedisease or condition, or to partially or fully reverse the effects ofthe disease.

By “prevent” is meant to minimize the chance that a subject who has anincreased susceptibility for developing a cancer will develop a cancer.

By “specifically binds” is meant that an antibody recognizes andphysically interacts with its cognate antigen (for example, a c-CBLpolypeptide) and does not significantly recognize and interact withother antigens; such an antibody may be a polyclonal antibody or amonoclonal antibody, which are generated by techniques that are wellknown in the art.

By “probe,” “primer,” or oligonucleotide is meant a single-stranded DNAor RNA molecule of defined sequence that can base-pair to a second DNAor RNA molecule that contains a complementary sequence (the “target”).The stability of the resulting hybrid depends upon the extent of thebase-pairing that occurs. The extent of base-pairing is affected byparameters such as the degree of complementarity between the probe andtarget molecules and the degree of stringency of the hybridizationconditions. The degree of hybridization stringency is affected byparameters such as temperature, salt concentration, and theconcentration of organic molecules such as formamide, and is determinedby methods known to one skilled in the art. Probes or primers specificfor c-CBL nucleic acids (for example, genes and/or mRNAs) have at least80%-90% sequence complementarity, preferably at least 91%-95% sequencecomplementarity, more preferably at least 96%-99% sequencecomplementarity, and most preferably 100% sequence complementarity tothe region of the c-CBL nucleic acid to which they hybridize. Probes,primers, and oligonucleotides may be detectably-labeled, eitherradioactively, or non-radioactively, by methods well-known to thoseskilled in the art. Probes, primers, and oligonucleotides are used formethods involving nucleic acid hybridization, such as: nucleic acidsequencing, reverse transcription and/or nucleic acid amplification bythe polymerase chain reaction, single stranded conformationalpolymorphism (SSCP) analysis, restriction fragment polymorphism (RFLP)analysis, Southern hybridization, Northern hybridization, in situhybridization, electrophoretic mobility shift assay (EMSA).

By “specifically hybridizes” is meant that a probe, primer, oroligonucleotide recognizes and physically interacts (that is,base-pairs) with a substantially complementary nucleic acid (forexample, a c-CBL nucleic acid) under high stringency conditions, anddoes not substantially base pair with other nucleic acids.

By “high stringency conditions” is meant conditions that allowhybridization comparable with that resulting from the use of a DNA probeof at least 40 nucleotides in length, in a buffer containing 0.5 MNaHPO₄, pH 7.2, 7% SDS, 1 mM EDTA, and 1% BSA (Fraction V), at atemperature of 65° C., or a buffer containing 48% formamide, 4.8×SSC,0.2 M Tris-Cl, pH 7.6, 1×Denhardt's solution, 10% dextran sulfate, and0.1% SDS, at a temperature of 42° C. Other conditions for highstringency hybridization, such as for PCR, Northern, Southern, or insitu hybridization, DNA sequencing, etc., are well-known by thoseskilled in the art of molecular biology. (See, for example, F. Ausubelet al., Current Protocols in Molecular Biology, John Wiley & Sons, NewYork, N.Y., 1998).

By “familial mutation” or “inherited mutation” is meant a mutation in anindividual that was inherited from a parent and that was present insomatic cells of the parent. By “sporadic mutation” or “spontaneousmutation” is meant a mutation in an individual that arose in theindividual and was not present in a parent of the individual.

As set forth herein, nucleotides are numbered according to the consensuscoding DNA sequence (CCDS) cDNA sequence for c-CBL (SEQ ID1 orCCDS8418), starting at nucleotide 1. The sequence is also set forth inTable 1.

The amino acid sequence of c-CBL are shown in SEQ ID2 or inUniProtKB/Swiss-Prot P22681, starting at amino acid 1, respectively. Thesequence is also set forth in Table 2.

TABLE 1 Consensus coding DNA sequence (CCDS) cDNA sequencefor c-CBL (SEQ ID NO: 1) (2721 nt)ATGGCCGGCAACGTGAAGAAGAGCTCTGGGGCCGGGGGCGGCAGCGGCTCCGGGGGCTCGGGTTCGGGTGGCCTGATTGGGCTCATGAAGGACGCCTTCCAGCCGCACCACCACCACCACCACCACCTCAGCCCCCACCCGCCGGGGACGGTGGACAAGAAGATGGTGGAGAAGTGCTGGAAGCTCATGGACAAGGTGGTGCGGTTGTGTCAGAACCCAAAGCTGGCGCTAAAGAATAGCCCACCTTATATCTTAGACCTGCTACCAGATACCTACCAGCATCTCCGTACTATCTTGTCAAGATATGAGGGGAAGATGGAGACACTTGGAGAAAATGAGTATTTTAGGGTGTTTATGGAGAATTTGATGAAGAAAACTAAGCAAACCATAAGCCTCTTCAAGGAGGGAAAAGAAAGAATGTATGAGGAGAATTCTCAGCCTAGGCGAAACCTAACCAAACTGTCCCTCATCTTCAGCCACATGCTGGCAGAACTAAAAGGAATCTTTCCAAGTGGACTCTTTCAGGGAGACACATTTCGGATTACTAAAGCAGATGCTGCGGAATTTTGGAGAAAAGCTTTTGGGGAAAAGACAATAGTCCCTTGGAAGAGCTTTCGACAGGCTCTACATGAAGTGCATCCCATCAGTTCTGGGCTGGAGGCCATGGCTCTGAAATCCACTATTGATCTGACCTGCAATGATTATATTTCGGTTTTTGAATTTGACATCTTTACCCGACTCTTTCAGCCCTGGTCCTCTTTGCTCAGGAATTGGAACAGCCTTGCTGTAACTCATCCTGGCTACATGGCTTTTTTGACGTATGACGAAGTGAAAGCTCGGCTCCAGAAATTCATTCACAAACCTGGCAGTTATATCTTCCGGCTGAGCTGTACTCGTCTGGGTCAGTGGGCTATTGGGTATGTTACTGCTGATGGGAACATTCTCCAGACAATCCCTCACAATAAACCTCTCTTCCAAGCACTGATTGATGGCTTCAGGGAAGGCTTCTATTTGTTTCCTGATGGACGAAATCAGAATCCTGATCTGACTGGCTTATGTGAACCAACTCCCCAAGACCATATCAAAGTGACCCAGGAACAATATGAATTATACTGTGAGATGGGCTCCACATTCCAACTATGTAAAATATGTGCTGAAAATGATAAGGATGTAAAGATTGAGCCCTGTGGACACCTCATGTGCACATCCTGTCTTACATCCTGGCAGGAATCAGAAGGTCAGGGCTGTCCTTTCTGCCGATGTGAAATTAAAGGTACTGAACCCATCGTGGTAGATCCGTTTGATCCTAGAGGGAGTGGCAGCCTGTTGAGGCAAGGAGCAGAGGGAGCTCCCTCCCCAAATTATGATGATGATGATGATGAACGAGCTGATGATACTCTCTTCATGATGAAGGAATTGGCTGGTGCCAAGGTGGAACGGCCGCCTTCTCCATTCTCCATGGCCCCACAAGCTTCCCTTCCCCCGGTGCCACCACGACTTGACCTTCTGCCGCAGCGAGTATGTGTTCCCTCAAGTGCTTCTGCTCTTGGAACTGCTTCTAAGGCTGCTTCTGGCTCCCTTCATAAAGACAAACCATTGCCAGTACCTCCCACACTTCGAGATCTTCCACCACCACCGCCTCCAGACCGGCCATATTCTGTTGGAGCAGAATCCCGACCTCAAAGACGCCCCTTGCCTTGTACACCAGGCGACTGTCCCTCCAGAGACAAACTGCCCCCTGTCCCCTCTAGCCGCCTTGGAGACTCATGGCTGCCCCGGCCAATCCCCAAAGTACCAGTATCTGCCCCAAGTTCCAGTGATCCCTGGACAGGAAGAGAATTAACCAACCGGCACTCACTTCCATTTTCATTGCCCTCACAAATGGAGCCCAGACCAGATGTGCCTAGGCTCGGAAGCACGTTCAGTCTGGATACCTCCATGAGTATGAATAGCAGCCCATTAGTAGGTCCAGAGTGTGACCACCCCAAAATCAAACCTTCCTCATCTGCCAATGCCATTTATTCTCTGGCTGCCAGACCTCTTCCTGTGCCAAAACTGCCACCTGGGGAGCAATGTGAGGGTGAAGAGGACACAAGTACATGACTCCCTCTTCCAGGCCTCTACGGCCTTTGGATACATCCCAGAGTTCACGAGCATGTGATTGCGACCAGCAGATTGATAGCTGTACGTATGAAGCAATGTATAATATTCAGTCCCAGGCGCCATCTATCACCGAGAGCAGCACCTTTGGTGAAGGGAATTTGGCCGCAGCCCATGCCAACACTGGTCCCGAGGAGTCAGAAAATGAGGATGATGGGTATGATGTCCCAAAGCCACCTGTGCCGGCCGTGCTGGCCCGCCGAACTCTCTCAGATATCTCTAATGCCAGCTCCTCCTTTGGCTGGTTGTCTCTGGATGGTGATCCTACAACAAATGTCACTGAAGGTTCCCAAGTTCCCGAGAGGCCTCCAAAACCATTCCCGCGGAGAATCAACTCTGAACGGAAAGCTGGCAGCTGTCAGCAAGGTAGTGGTCCTGCCGCCTCTGCTGCCACCGCCTCACCTCAGCTCTCCAGTGAGATCGAGAACCTCATGAGTCAGGGGTACTCCTACCAGGACATCCAGAAAGCTTTGGTCATTGCCCAGAACAACATCGAGATGGCCAAAAACATCCTCCGGGAATTTGTTTCCATTTCTTCTC CTGCCCATGTAGCTACCTAG

TABLE 2 SEQ ID NO: 2 Amino Acid Sequence (Amino Acids 1-906)MAGNVKKSSGAGGGSGSGGSGSGGLIGLMKDAFQPHHHHHHHLSPHPPGTVDKKMVEKCWKLMDKVVRLCQNPKLALKNSPPYILDLLPDTYQHLRTILSRYEGKMETLGENEYFRVFMENLMKKTKQTISLFKEGKERMYEENSQPRRNLTKLSLIFSHMLAELKGIFPSGLFQGDTFRITKADAAEFWRKAFGEKTIVPWKSFRQALHEVHPISSGLEAMALKSTIDLTCNDYISVFEFDIFTRLFQPWSSLLRNWNSLAVTHPGYMAFLTYDEVKARLQKFIHKPGSYIFRLSCTRLGQWAIGYVTADGNILQTIPHNKPLFQALIDGFREGFYLFPDGRNQNPDLTGLCEPTPQDHIKVTQEQYELYCEMGSTFQLCKICAENDKDVKIEPCGHLMCTSCLTSWQESEGQGCPFCRCEIKGTEPIVVDPFDPRGSGSLLRQGAEGAPSPNYDDDDDERADDTLFMMKELAGAKVERPPSPFSMAPQASLPPVPPRLDLLPQRVCVPSSASALGTASKAASGSLHKDKPLPVPPTLRDLPPPPPPDRPYSVGAESRPQRRPLPCTPGDCPSRDKLPPVPSSRLGDSWLPRPIPKVPVSAPSSSDPWTGRELTNRHSLPFSLPSQMEPRPDVPRLGSTFSLDTSMSMNSSPLVGPECDHPKIKPSSSANAIYSLAARPLPVPKLPPGEQCEGEEDTEYMTPSSRPLRPLDTSQSSRACDCDQQIDSCTYEAMYNIQSQAPSITESSTFGEGNLAAAHANTGPEESENEDDGYDVPKPPVPAVLARRTLSDISNASSSFGWLSLDGDPTTNVTEGSQVPERPPKPFPRRINSERKAGSCQQGSGPAASAATASPQLSSEIENLMSQGYSYQDIQKALVIAQNNIEMAKNILREFVSISS PAHVAT

As used herein, a specific notation will be used to denote certain typesof mutations. All notations referencing a nucleotide or amino acidresidue will be understood to correspond to the residue number of thewild-type c-CBL nucleic acid sequence set forth at SEQ ID NO:1, or ofthe wild-type c-CBL polypeptide sequence set forth at SEQ ID NO:2. Thus,for example, the notation “S80N” when used in the context of apolypeptide sequence will be used to indicate that the amino acid Serineat position 80 has been replaced with Asparagine.

In the method of the invention, the mutant c-CBL polypeptide or mutatedc-CBL nucleic acid identified can be associated with cancers.

Compositions

The disclosed compositions are related to c-CBL. Disclosed herein arecompositions, such as polynucleotides capable of specificallyhybridizing to c-CBL encoding human c-CBL, wherein the mutation resultsin an amino acid change at position S80N, H94Y, Q249E, V391I,72515_72517delATG, R830K, A848T, L620F, P170L, S171S, L281F, L254S, orP782L, or a stop codon at position W802*, or a complement thereof.

Also disclosed are isolated polynucleotides that comprise mutations in anucleotide sequence capable of encoding a c-CBL protein, that do notresult in a change in the amino acid sequence. Such mutations cansometimes be referred to as “silent mutations”. “Silent mutations”described above, can be used in the same methods and within the samecompositions as the other mutations described herein.

The disclosed nucleic acids are made up of for example, nucleotides,nucleotide analogs, or nucleotide substitutes. Non-limiting examples ofthese and other molecules are discussed herein. It is understood thatfor example, when a vector is expressed in a cell that the expressedmRNA will typically be made up of A, C, G, and U. Likewise, it isunderstood that if, for example, an antisense molecule is introducedinto a cell or cell environment through for example exogenous delivery,it is advantageous that the antisense molecule be made up of nucleotideanalogs that reduce the degradation of the antisense molecule in thecellular environment.

The nucleotides of the invention can comprise one or more nucleotideanalogs or substitutions. A nucleotide analog is a nucleotide whichcontains some type of modification to either the base, sugar, orphosphate moieties. Modifications to the base moiety would includenatural and synthetic modifications of A, C, G, and T/U as well asdifferent purine or pyrimidine bases, such as uracil-5-yl (ψ),hypoxanthin-9-yl (I), and 2-aminoadenin-9-yl. A modified base includesbut is not limited to 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and otheralkyl derivatives of adenine and guanine, 2-propyl and other alkylderivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil andcytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil),4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl andother 8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine,7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.Additional base modifications can be found for example in U.S. Pat. No.3,687,808, Englisch et al., Angewandte Chemie, International Edition,1991, 30, 613, and Sanghvi, Y. S., Chapter 15, Antisense Research andApplications, pages 289-302, Crooke, S. T. and Lebleu, B. ed., CRCPress, 1993. Certain nucleotide analogs, such as 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine can increase the stability of duplex formation. Oftentime base modifications can be combined with for example a sugarmodification, such as 2′-O-methoxyethyl, to achieve unique propertiessuch as increased duplex stability. There are numerous United Statespatents such as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066;5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908;5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091;5,614,617; and 5,681,941, which detail and describe a range of basemodifications. Each of these patents is herein incorporated byreference.

Nucleotide analogs can also include modifications of the sugar moiety.Modifications to the sugar moiety would include natural modifications ofthe ribose and deoxy ribose as well as synthetic modifications. Sugarmodifications include but are not limited to the following modificationsat the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-,S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl andalkynyl may be substituted or unsubstituted C₁ to C₁₀, alkyl or C₂ toC₁₀ alkenyl and alkynyl. 2′ sugar modifications also include but are notlimited to —O[(CH₂)_(n) 0]. CH₃, —O(CH₂)_(n) OCH₃, —O(CH₂)_(n) NH₂,—O(CH₂)_(n) CH₃, —O(CH₂)_(n) —ONH₂, and —O(CH₂)_(n)ON[(CH₂)_(n) CH₃)]₂,where n and m are from 1 to about 10.

Other modifications at the 2′ position include but are not limited to:C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl,O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacokinetic properties of an oligonucleotide, or a group forimproving the pharmacodynamic properties of an oligonucleotide, andother substituents having similar properties. Similar modifications mayalso be made at other positions on the sugar, particularly the 3′position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide. Modifiedsugars would also include those that contain modifications at thebridging ring oxygen, such as CH₂ and S. Nucleotide sugar analogs mayalso have sugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. There are numerous United States patents thatteach the preparation of such modified sugar structures such as U.S.Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265;5,658,873; 5,670,633; and 5,700,920, each of which is hereinincorporated by reference in its entirety for their teaching ofmodifications and methods related to the same.

Nucleotide analogs can also be modified at the phosphate moiety.Modified phosphate moieties include but are not limited to those thatcan be modified so that the linkage between two nucleotides contains aphosphorothioate, chiral phosphorothioate, phosphorodithioate,phosphotriester, aminoalkylphosphotriester, methyl and other alkylphosphonates including 3′-alkylene phosphonate and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates. It is understood that these phosphate or modifiedphosphate linkage between two nucleotides can be through a 3′-5′ linkageor a 2′-5′ linkage, and the linkage can contain inverted polarity suchas 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and freeacid forms are also included. Numerous United States patents teach howto make and use nucleotides containing modified phosphates and includebut are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301;5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302;5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233;5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111;5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of which is hereinincorporated by reference in its entirety for their teaching ofmodifications and methods related to the same.

Nucleotide substitutes are molecules having similar functionalproperties to nucleotides, but which do not contain a phosphate moiety,such as peptide nucleic acid (PNA). Nucleotide substitutes are moleculesthat will recognize nucleic acids in a Watson-Crick or Hoogsteen manner,but which are linked together through a moiety other than a phosphatemoiety. Nucleotide substitutes are able to conform to a double helixtype structure when interacting with the appropriate target nucleicacid.

Nucleotide substitutes are nucleotides or nucleotide analogs that havehad the phosphate moiety or sugar moieties replaced. Nucleotidesubstitutes do not contain a standard phosphorus atom. Substitutes forthe phosphate can be, for example, short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatom and alkyl or cycloalkylinternucleoside linkages, or one or more short chain heteroatomic orheterocyclic internucleoside linkages. These include those havingmorpholino linkages (formed in part from the sugar portion of anucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts. Numerous United States patents disclosehow to make and use these types of phosphate replacements and includebut are not limited to U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444;5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938;5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225;5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289;5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439,each of which is herein incorporated by reference in its entirety fortheir teaching of modifications and methods related to the same.

It is also understood in a nucleotide substitute that both the sugar andthe phosphate moieties of the nucleotide can be replaced, by for examplean amide type linkage (aminoethylglycine) (PNA). U.S. Pat. Nos.5,539,082; 5,714,331; and 5,719,262 teach how to make and use PNAmolecules, each of which is herein incorporated by reference in itsentirety for their teaching of modifications and methods related to thesame. (See also Nielsen et al., Science, 254, 1497-1500 (1991)).

It is also possible to link other types of molecules (conjugates) tonucleotides or nucleotide analogs to enhance for example, cellularuptake. Conjugates can be chemically linked to the nucleotide ornucleotide analogs. Such conjugates include but are not limited to lipidmoieties such as a cholesterol moiety (Letsinger et al., Proc. Natl.Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al.,Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g.,hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660,306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770),a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20,533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues(Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al.,FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75,49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al.,Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethyleneglycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14,969-973), or adamantane acetic acid (Manoharan et al., TetrahedronLett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim.Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937.

Numerous United States patents teach the preparation of such conjugatesand include, but are not limited to U.S. Pat. Nos. 4,828,979; 4,948,882;5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717,5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045;5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044;4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263;4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136;5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506;5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723;5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552;5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696;5,599,923; 5,599,928 and 5,688,941, each of which is herein incorporatedby reference in its entirety for their teaching of modifications andmethods related to the same.

The same methods of calculating homology as described elsewhere hereinconcerning polypeptides can be obtained for nucleic acids by for examplethe algorithms disclosed in Zuker, M. Science 244:48-52, 1989, Jaeger etal. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, and Jaeger et al.Methods Enzymol. 183:281-306, 1989 which are herein incorporated byreference for at least material related to nucleic acid alignment.

Also, disclosed are compositions including primers and probes, which arecapable of interacting with the polynucleotide sequences disclosedherein. For example, disclosed are primers/probes capable of amplifyinga nucleic acid encoding human c-CBL, wherein the mutation results in anamino acid change at position S80N, H94Y, Q249E, V391I,72515_72517delATG, W802*, R830K, A848T, L620F, P170L, S171S, L281F,L254S, or P782L. Examples of such primers/probes are disclosed elsewhereherein. For example, examples of primers and probes can be found in theExamples section below.

The disclosed primers can used to support DNA amplification reactions.Typically the primers will be capable of being extended in a sequencespecific manner. Extension of a primer in a sequence specific mannerincludes any methods wherein the sequence or composition of the nucleicacid molecule to which the primer is hybridized or otherwise associateddirects or influences the composition or sequence of the productproduced by the extension of the primer. Extension of the primer in asequence specific manner therefore includes, but is not limited to, PCR,DNA sequencing, DNA extension, DNA polymerization, RNA transcription, orreverse transcription. Techniques and conditions that amplify the primerin a sequence specific manner are preferred. In certain embodiments theprimers are used for the DNA amplification reactions, such as PCR ordirect sequencing. It is understood that in certain embodiments theprimers can also be extended using non-enzymatic techniques, where forexample, the nucleotides or oligonucleotides used to extend the primerare modified such that they will chemically react to extend the primerin a sequence specific manner. Typically the disclosed primers hybridizewith the polynucleotide sequences disclosed herein or region of thepolynucleotide sequences disclosed herein or they hybridize with thecomplement of the polynucleotide sequences disclosed herein orcomplement of a region of the polynucleotide sequences disclosed herein.

The size of the primers or probes for interaction with thepolynucleotide sequences disclosed herein in certain embodiments can beany size that supports the desired enzymatic manipulation of the primer,such as DNA amplification or the simple hybridization of the probe orprimer. A typical primer or probe would be at least 6, 7, 8, 9, 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300,325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800,850, 900, 950, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000,3500, or 4000 nucleotides long or any length in-between.

Also disclosed is an isolated polynucleotide capable of distinguishingbetween isolated polynucleotides capable of encoding polypeptidescomprising a mutation at a nucleic acid position corresponding to anamino acid change at position S80N, H94Y, Q249E, V391I,72515_72517delATG, W802*, R830K, A848T, L620F, P170L, S171S, L281F,L254S, or P782L, or a complement thereof, and a nucleic acid encoding awild type c-CBL.

Optionally, isolated polypeptides or isolated nucleotides can also bepurified, e.g., are at least about 90% pure, more preferably at leastabout 95% pure and most preferably at least about 99% pure.

Also disclosed are the components to be used to prepare the disclosedcompositions as well as the compositions themselves to be used withinthe methods disclosed herein. These and other materials are disclosedherein, and it is understood that when combinations, subsets,interactions, groups, etc. of these materials are disclosed that whilespecific reference of each various individual and collectivecombinations and permutation of these compounds may not be explicitlydisclosed, each is specifically contemplated and described herein. Forexample, if a particular polypeptide is disclosed and discussed and anumber of modifications that can be made to a number of moleculesincluding the polypeptide are discussed, specifically contemplated areeach and every combination and permutation of polypeptide and themodifications that are possible unless specifically indicated to thecontrary. Thus, if a class of molecules A, B, and C are disclosed aswell as a class of molecules D, E, and F and an example of a combinationmolecule, A-D is disclosed, then even if each is not individuallyrecited each is individually and collectively contemplated meaningcombinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considereddisclosed. Likewise, any subset or combination of these is alsodisclosed. Thus, for example, the sub-group of A-E, B-F, and C-E wouldbe considered disclosed. This concept applies to all aspects of thisapplication including, but not limited to, steps in methods of makingand using the disclosed compositions. Thus, if there are a variety ofadditional steps that can be performed it is understood that each ofthese additional steps can be performed with any specific embodiment orcombination of embodiments of the disclosed methods.

It is understood that one way to define any known variants andderivatives or those that might arise, of the disclosed genes andproteins herein is through defining the variants and derivatives interms of homology to specific known sequences. For example SEQ ID NO: 1sets forth a particular sequence of the wild-type c-CBL gene and SEQ IDNO: 2 sets forth a particular sequence of the protein encoded by SEQ IDNO: 1. Specifically disclosed are variants of these and other genes andproteins herein disclosed which have at least, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, or 99 percent homology to the stated sequence. Thoseof skill in the art readily understand how to determine the homology oftwo proteins or nucleic acids, such as genes. For example, the homologycan be calculated after aligning the two sequences so that the homologyis at its highest level.

Another way of calculating homology can be performed by publishedalgorithms. Optimal alignment of sequences for comparison may beconducted by the local homology algorithm of Smith and Waterman Adv.Appl. Math. 2: 482 (1981), by the homology alignment algorithm ofNeedleman and Wunsch, J. MoL Biol. 48: 443 (1970), by the search forsimilarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85: 2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or byinspection.

The same types of homology can be obtained for nucleic acids by forexample the algorithms disclosed in Zuker, M. Science 244:48-52, 1989,Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger etal. Methods Enzymol. 183:281-306, 1989 which are herein incorporated byreference for at least material related to nucleic acid alignment.

For example, as used herein, a sequence recited as having a particularpercent homology to another sequence refers to sequences that have therecited homology as calculated by any one or more of the calculationmethods described above. For example, a first sequence has 80 percenthomology, as defined herein, to a second sequence if the first sequenceis calculated to have 80 percent homology to the second sequence usingthe Zuker calculation method even if the first sequence does not have 80percent homology to the second sequence as calculated by any of theother calculation methods. As another example, a first sequence has 80percent homology, as defined herein, to a second sequence if the firstsequence is calculated to have 80 percent homology to the secondsequence using both the Zuker calculation method and the Pearson andLipman calculation method even if the first sequence does not have 80percent homology to the second sequence as calculated by the Smith andWaterman calculation method, the Needleman and Wunsch calculationmethod, the Jaeger calculation methods, or any of the other calculationmethods. As yet another example, a first sequence has 80 percenthomology, as defined herein, to a second sequence if the first sequenceis calculated to have 80 percent homology to the second sequence usingeach of calculation methods (although, in practice, the differentcalculation methods will often result in different calculated homologypercentages).

The term hybridization typically means a sequence driven interactionbetween at least two nucleic acid molecules, such as a primer or a probeand a gene. Sequence driven interaction means an interaction that occursbetween two nucleotides or nucleotide analogs or nucleotide derivativesin a nucleotide specific manner. For example, G interacting with C or Ainteracting with T are sequence driven interactions. Typically sequencedriven interactions occur on the Watson-Crick face or Hoogsteen face ofthe nucleotide. The hybridization of two nucleic acids is affected by anumber of conditions and parameters known to those of skill in the art.For example, the salt concentrations, pH, and temperature of thereaction all affect whether two nucleic acid molecules will hybridize.

Parameters for selective hybridization between two nucleic acidmolecules are well known to those of skill in the art. For example, insome embodiments selective hybridization conditions can be defined asstringent hybridization conditions. For example, stringency ofhybridization is controlled by both temperature and salt concentrationof either or both of the hybridization and washing steps. For example,the conditions of hybridization to achieve selective hybridization mayinvolve hybridization in high ionic strength solution (6×SSC or 6×SSPE)at a temperature that is about 12-25° C. below the Tm (the meltingtemperature at which half of the molecules dissociate from theirhybridization partners) followed by washing at a combination oftemperature and salt concentration chosen so that the washingtemperature is about 5° C. to 20° C. below the Tm. The temperature andsalt conditions are readily determined empirically in preliminaryexperiments in which samples of reference DNA immobilized on filters arehybridized to a labeled nucleic acid of interest and then washed underconditions of different stringencies. Hybridization temperatures aretypically higher for DNA-RNA and RNA-RNA hybridizations. The conditionscan be used as described above to achieve stringency, or as is known inthe art. (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ndEd., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989;Kunkel et al. Methods Enzymol. 1987:154:367, 1987 which is hereinincorporated by reference in its entirety and at least for materialrelated to hybridization of nucleic acids). As used herein “stringenthybridization” for a DNA:DNA hybridization is about 68° C. (in aqueoussolution) in 6×SSC or 6×SSPE followed by washing at 68° C. Stringency ofhybridization and washing, if desired, can be reduced accordingly as thedegree of complementarity desired is decreased, and further, dependingupon the G-C or A-T richness of any area wherein variability is searchedfor. Likewise, stringency of hybridization and washing, if desired, canbe increased accordingly as homology desired is increased, and further,depending upon the G-C or A-T richness of any area wherein high homologyis desired, all as known in the art.

Another way to define selective hybridization is by looking at theamount (percentage) of one of the nucleic acids bound to the othernucleic acid. For example, in some embodiments selective hybridizationconditions would be when at least about, 60, 65, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100 percent of the limiting nucleic acid isbound to the non-limiting nucleic acid. Typically, the non-limitingprimer is in for example, 10 or 100 or 1000 fold excess. This type ofassay can be performed at under conditions where both the limiting andnon-limiting primer are for example, 10 fold or 100 fold or 1000 foldbelow their k_(d), or where only one of the nucleic acid molecules is 10fold or 100 fold or 1000 fold or where one or both nucleic acidmolecules are above their k_(d).

Another way to define selective hybridization is by looking at thepercentage of primer that gets enzymatically manipulated underconditions where hybridization is required to promote the desiredenzymatic manipulation. For example, in some embodiments selectivehybridization conditions would be when at least about, 60, 65, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the primer isenzymatically manipulated under conditions which promote the enzymaticmanipulation, for example if the enzymatic manipulation is DNAextension, then selective hybridization conditions would be when atleast about 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100percent of the primer molecules are extended. Preferred conditions alsoinclude those suggested by the manufacturer or indicated in the art asbeing appropriate for the enzyme performing the manipulation.

Just as with homology, it is understood that there are a variety ofmethods herein disclosed for determining the level of hybridizationbetween two nucleic acid molecules. It is understood that these methodsand conditions may provide different percentages of hybridizationbetween two nucleic acid molecules, but unless otherwise indicatedmeeting the parameters of any of the methods would be sufficient. Forexample if 80% hybridization was required and as long as hybridizationoccurs within the required parameters in any one of these methods it isconsidered disclosed herein.

It is understood that those of skill in the art understand that if acomposition or method meets any one of these criteria for determininghybridization either collectively or singly it is a composition ormethod that is disclosed herein. Optionally, one or more of the isolatedpolynucleotides of the invention are attached to a solid support. Solidsupports are disclosed herein.

Also disclosed herein are arrays comprising polynucleotides capable ofspecifically hybridizing to c-CBL encoding nucleic acid comprising amutation at a nucleic acid position corresponding to a change in aminoacid at position S80N, H94Y, Q249E, V391I, 72515_72517delATG, W802*,R830K, A848T, L620F, P170L, S171S, L281F, L254S, or P782L. Alsodisclosed are arrays comprising polynucleotides capable of specificallyhybridizing to c-CBL encoding nucleic acids that comprise a mutation ina nucleic acid sequence encoding human c-CBL, wherein the mutation islocated in the TKB domain, RING finger domain, proline-rich region,C-terminal region, or other domain linkage regions of c-CBL. Alsodisclosed herein are solid supports comprising one or more of thedisclosed polynucleotides or polypeptides capable of hybridizing to amutant form of c-CBL.

Solid supports are solid-state substrates or supports with whichmolecules, such as analytes and analyte binding molecules, can beassociated. Analytes, such as calcifying nano-particles and proteins,can be associated with solid supports directly or indirectly. Forexample, analytes can be directly immobilized on solid supports. Analytecapture agents, such a capture compounds, can also be immobilized onsolid supports. For example, disclosed herein are antigen binding agentscapable of specifically binding to a c-CBL polypeptides comprising amutation at position S80N, H94Y, Q249E, V391I, 72515_72517delATG, W802*,R830K, A848T, L620F, P170L, S171S, L281F, L254S, or P782L. Alsodisclosed is an antigen binding agent capable of specifically binding toa c-CBL polypeptide comprising a mutation, wherein the mutation islocated in the TKB domain, RING finger domain, proline-rich region,C-terminal region, or other domain linkage regions of c-CBL.

A preferred form of solid support is an array. Another form of solidsupport is an array detector. An array detector is a solid support towhich multiple different capture compounds or detection compounds havebeen coupled in an array, grid, or other organized pattern.

Solid-state substrates for use in solid supports can include any solidmaterial to which molecules can be coupled. This includes materials suchas acrylamide, agarose, cellulose, nitrocellulose, glass, polystyrene,polyethylene vinyl acetate, polypropylene, polymethacrylate,polyethylene, polyethylene oxide, polysilicates, polycarbonates, teflon,fluorocarbons, nylon, silicon rubber, polyanhydrides, polyglycolic acid,polylactic acid, polyorthoesters, polypropylfumerate, collagen,glycosaminoglycans, and polyamino acids. Solid-state substrates can haveany useful form including thin film, membrane, bottles, dishes, fibers,woven fibers, shaped polymers, particles, beads, microparticles, or acombination. Solid-state substrates and solid supports can be porous ornon-porous. A preferred form for a solid-state substrate is a microtiterdish, such as a standard 96-well type. In preferred embodiments, amultiwell glass slide can be employed that normally contain one arrayper well. This feature allows for greater control of assayreproducibility, increased throughput and sample handling, and ease ofautomation.

Different compounds can be used together as a set. The set can be usedas a mixture of all or subsets of the compounds used separately inseparate reactions, or immobilized in an array. Compounds usedseparately or as mixtures can be physically separable through, forexample, association with or immobilization on a solid support. An arraycan include a plurality of compounds immobilized at identified orpredefined locations on the array. Each predefined location on the arraygenerally can have one type of component (that is, all the components atthat location are the same). Each location will have multiple copies ofthe component. The spatial separation of different components in thearray allows separate detection and identification of thepolynucleotides or polypeptides disclosed herein.

Although preferred, it is not required that a given array be a singleunit or structure. The set of compounds may be distributed over anynumber of solid supports. For example, at one extreme, each compound maybe immobilized in a separate reaction tube or container, or on separatebeads or microparticles. Different modes of the disclosed method can beperformed with different components (for example, different compoundsspecific for different proteins) immobilized on a solid support.

Some solid supports can have capture compounds, such as antibodies,attached to a solid-state substrate. Such capture compounds can bespecific for calcifying nano-particles or a protein on calcifyingnano-particles. Captured calcifying nano-particles or proteins can thenbe detected by binding of a second, detection compound, such as anantibody. The detection compound can be specific for the same or adifferent protein on the calcifying nano-particle.

Methods for immobilizing antibodies (and other proteins) to solid-statesubstrates are well established. Immobilization can be accomplished byattachment, for example, to aminated surfaces, carboxylated surfaces orhydroxylated surfaces using standard immobilization chemistries.Examples of attachment agents are cyanogen bromide, succinimide,aldehydes, tosyl chloride, avidin-biotin, photocrosslinkable agents,epoxides and maleimides. A preferred attachment agent is theheterobifunctional cross-linker N-[γ-Maleimidobutyryloxy] succinimideester (GMBS). These and other attachment agents, as well as methods fortheir use in attachment, are described in Protein immobilization:fundamentals and applications, Richard F. Taylor, ed. (M. Dekker, NewYork, 1991); Johnstone and Thorpe, Immunochemistry In Practice(Blackwell Scientific Publications, Oxford, England, 1987) pages 209-216and 241-242, and Immobilized Affinity Ligands; Craig T. Hermanson etal., eds. (Academic Press, New York, 1992) which are incorporated byreference in their entirety for methods of attaching antibodies to asolid-state substrate. Antibodies can be attached to a substrate bychemically cross-linking a free amino group on the antibody to reactiveside groups present within the solid-state substrate. For example,antibodies may be chemically cross-linked to a substrate that containsfree amino, carboxyl, or sulfur groups using glutaraldehyde,carbodiimides, or GMBS, respectively, as cross-linker agents. In thismethod, aqueous solutions containing free antibodies are incubated withthe solid-state substrate in the presence of glutaraldehyde orcarbodiimide.

A preferred method for attaching antibodies or other proteins to asolid-state substrate is to functionalize the substrate with an amino-or thiol-silane, and then to activate the functionalized substrate witha homobifunctional cross-linker agent such as (Bis-sulfo-succinimidylsuberate (BS³) or a heterobifunctional cross-linker agent such as GMBS.For cross-linking with GMBS, glass substrates are chemicallyfunctionalized by immersing in a solution ofmercaptopropyltrimethoxysilane (1% vol/vol in 95% ethanol pH 5.5) for 1hour, rinsing in 95% ethanol and heating at 120° C. for 4 hrs.Thiol-derivatized slides are activated by immersing in a 0.5 mg/mlsolution of GMBS in 1% dimethylformamide, 99% ethanol for 1 hour at roomtemperature. Antibodies or proteins are added directly to the activatedsubstrate, which are then blocked with solutions containing agents suchas 2% bovine serum albumin, and air-dried. Other standard immobilizationchemistries are known by those of skill in the art.

Each of the components (compounds, for example) immobilized on the solidsupport preferably is located in a different predefined region of thesolid support. Each of the different predefined regions can bephysically separated from each other of the different regions. Thedistance between the different predefined regions of the solid supportcan be either fixed or variable. For example, in an array, each of thecomponents can be arranged at fixed distances from each other, whilecomponents associated with beads will not be in a fixed spatialrelationship. In particular, the use of multiple solid support units(for example, multiple beads) will result in variable distances.

Components can be associated or immobilized on a solid support at anydensity. Components preferably are immobilized to the solid support at adensity exceeding 400 different components per cubic centimeter. Arraysof components can have any number of components. For example, an arraycan have at least 1,000 different components immobilized on the solidsupport, at least 10,000 different components immobilized on the solidsupport, at least 100,000 different components immobilized on the solidsupport, or at least 1,000,000 different components immobilized on thesolid support.

Optionally, at least one address on the solid support is the sequencesor part of the sequences set forth in any of the nucleic acid sequencesdisclosed herein. Also disclosed are solid supports where at least oneaddress is the sequences or portion of sequences set forth in any of thepeptide sequences disclosed herein. Solid supports can also contain atleast one address is a variant of the sequences or part of the sequencesset forth in any of the nucleic acid sequences disclosed herein. Solidsupports can also contain at least one address is a variant of thesequences or portion of sequences set forth in any of the peptidesequences disclosed herein.

Also disclosed are antigen microarrays for multiplex characterization ofantibody responses. For example, disclosed are antigen arrays andminiaturized antigen arrays to perform large-scale multiplexcharacterization of antibody responses directed against thepolypeptides, polynucleotides and antibodies described herein, usingsubmicroliter quantities of biological samples as described in Robinsonet al., Autoantigen microarrays for multiplex characterization ofautoantibody responses, Nat Med., 8(3):295-301 (2002), which in hereinincorporated by reference in its entirety for its teaching ofcontructing and using antigen arrays to perform large-scale multiplexcharacterization of antibody responses directed against structurallydiverse antigens, using submicroliter quantities of biological samples.

Protein variants and derivatives are well understood to those of skillin the art and can involve amino acid sequence modifications. Forexample, amino acid sequence modifications typically fall into one ormore of three classes: substitutional, insertional or deletionalvariants. Polypeptide variants generally encompassed by the presentinvention will typically exhibit at least about 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity(determined as described below), along its length, to a polypeptidesequences set forth herein.

Also disclosed are expression vectors comprising the polynucleotidesdescribed elsewhere herein. For example, disclosed are expressionvectors comprising the polynucleotides described elsewhere herein,operably linked to a control element. Also disclosed herein are hostcells transformed or transfected with an expression vector comprisingthe polynucleotides described elsewhere herein. Also disclosed are hostcells comprising the expression vectors described herein. For example,disclosed is a host cell comprising an expression vector comprising thepolynucleotides described elsewhere herein, operably linked to a controlelement. Host cells can be eukayotic or prokaryotic cells.

Expression vectors can be any nucleotide construction used to delivergenes into cells (e.g., a plasmid), or as part of a general strategy todeliver genes, e.g., as part of recombinant retrovirus or adenovirus(Ram et al. Cancer Res. 53:83-88, (1993)). For example, disclosed hereinare expression vectors comprising an isolated polynucleotide comprisinga sequence of one or more of the c-CBL mutants described herein,operably linked to a control element.

As used herein, plasmid or viral vectors are agents that transport thedisclosed nucleic acids, such as an isolated polynucleotide capable ofencoding one or more polypeptides disclosed herein into the cell withoutdegradation and include a promoter yielding expression of the gene inthe cells into which it is delivered. In some embodiments the isolatedpolynucleotides disclosed herein are derived from either a virus or aretrovirus.

As described herein, the compositions can be administered in apharmaceutically acceptable carrier and can be delivered to thesubject's cells in vivo or ex vivo by a variety of mechanisms well knownin the art (e.g., uptake of naked DNA, liposome fusion, intramuscularinjection of DNA via a gene gun, endocytosis and the like).

If ex vivo methods are employed, cells or tissues can be removed andmaintained outside the body according to standard protocols well knownin the art. The compositions can be introduced into the cells via anygene transfer mechanism, such as, for example, calcium phosphatemediated gene delivery, electroporation, microinjection orproteoliposomes. The transduced cells can then be infused (e.g., in apharmaceutically acceptable carrier) or homotopically transplanted backinto the subject per standard methods for the cell or tissue type.Standard methods are known for transplantation or infusion of variouscells into a subject.

The nucleic acids, such as, the polynucleotides described herein, can bemade using standard chemical synthesis methods or can be produced usingenzymatic methods or any other known method. Such methods can range fromstandard enzymatic digestion followed by nucleotide fragment isolation(see for example, Sambrook et al., Molecular Cloning: A LaboratoryManual, 3rd Edition (Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 2001) Chapters 5, 6) to purely synthetic methods, forexample, by the cyanoethyl phosphoramidite method using a Milligen orBeckman System 1Plus DNA synthesizer. Synthetic methods useful formaking oligonucleotides are also described by Ikuta et al., Ann. Rev.Biochem. 53:323-356 (1984), (phosphotriester and phosphite-triestermethods), and Narang et al., Methods Enzymol., 65:610-620 (1980),(phosphotriester method). Protein nucleic acid molecules can be madeusing known methods such as those described by Nielsen et al.,Bioconjug. Chem. 5:3-7 (1994).

The invention also provides polypeptides related to c-CBL. As usedherein, the term “polypeptide” is used in its conventional meaning,i.e., as a sequence of amino acids. The polypeptides are not limited toa specific length of the product; thus, peptides, oligopeptides, andproteins are included within the definition of polypeptide, and suchterms may be used interchangeably herein unless specifically indicatedotherwise. This term also does not refer to or exclude post-expressionmodifications of the polypeptide, for example, glycosylations,acetylations, phosphorylations and the like, as well as othermodifications known in the art, both naturally occurring andnon-naturally occurring. A polypeptide may be an entire protein, or asubsequence thereof. Particular polypeptides of interest in the contextof this invention are amino acid subsequences comprising epitopes, i.e.,antigenic determinants substantially responsible for the immunogenicproperties of a polypeptide and being capable of evoking an immuneresponse.

Thus, a “c-CBL polypeptide” or “c-CBL protein,” refers generally to apolypeptide sequence that is present in samples isolated from normalsubjects as well as a substantial proportion of subjects with a cancer,for example preferably greater than about 20%, more preferably greaterthan about 30%, and most preferably greater than about 50% or more ofsubjects tested as determined using a representative assay providedherein. A polypeptide sequence of the invention, based upon itsexpression in a cancer sample isolated from individuals with a cancer,has particular utility both as a diagnostic, prognostic and/ortheranostic marker as well as a therapeutic target, as further describedbelow.

For example, disclosed herein are polypeptides comprising an amino acidsequence encoded by the polynucleotides described elsewhere herein. Forexample, disclosed are isolated polypeptides comprising amino acidsequences disclosed herein. Also disclosed are isolated polynucleotidescapable of encoding one or more polypeptides selected from the groupconsisting of the polypeptides disclosed herein, or a complementthereof.

The polypeptides of the present invention are sometimes herein referredto as c-CBL proteins or c-CBL polypeptides, as an indication that theiridentification has been based at least in part upon their expression incancer samples isolated from tissues of a subject with lung cancer orhead and neck cancer. The peptides described herein are identified fromtissues for a subject with either lung cancer and head and neck cancer.Accordingly, such a peptide may not be present in adjacent normaltissue. However, non-mutant forms of the polynucleotides andpolypeptides can be found in normal tissue.

Additionally, polypeptides described herein may be identified by theirdifferent reactivity with sera from subjects with cancer as compared tosera from unaffected individuals. For example, polypeptides describedherein may be identified by their reactivity with sera from subjectswith a cancer as compared to their lack of reactivity to sera fromunaffected individuals. Additionally, polypeptides described herein maybe identified by their reactivity with sera from subjects with cancer ascompared to their higher reactivity to sera from unaffected individuals.Additionally, polypeptides described herein may be identified by theirreactivity with sera from subjects with a cancer as compared to theirlower reactivity to sera from unaffected individuals.

Also disclosed herein are antigen binding agents capable of specificallybinding to a c-CBL polypeptide comprising a mutation at position S80N,H94Y, Q249E, V391I, 72515_72517delATG, W802*, R830K, A848T, L620F,P170L, S171S, L281F, L254S, or P782L. Also disclosed is an antigenbinding agent capable of specifically binding to a c-Met polypeptidecomprising a mutation in a nucleic acid sequence encoding human c-CBL,wherein the mutation is located in the TKB domain, RING finger domain,proline-rich region, C-terminal region, or other domain linkage regionsof c-CBL.

Also disclosed are isolated polypeptides comprising the sequenceprovided herein as well as the Figures, with substituted, inserted ordeletional variations.

As this specification discusses various polypeptides and polypeptidesequences it is understood that the nucleic acids that can encode thosepolypeptide sequences are also disclosed. This would include alldegenerate sequences related to a specific polypeptide sequence, i.e.all nucleic acids having a sequence that encodes one particularpolypeptide sequence as well as all nucleic acids, including degeneratenucleic acids, encoding the disclosed variants and derivatives of theprotein sequences. Thus, while each particular nucleic acid sequence maynot be written out herein, it is understood that each and every sequenceis in fact disclosed and described herein through the disclosedpolypeptide sequences.

Also disclosed herein are isolated antibodies, antibody fragments andantigen-binding fragments thereof, that specifically bind to apolypeptide sequence described herein. Optionally, the isolatedantibodies, antibody fragments, or antigen-binding fragment thereof canbe neutralizing antibodies. The antibodies, antibody fragments andantigen-binding fragments thereof disclosed herein can be identifiedusing the methods disclosed herein. For example, antibodies that bind tothe polypeptides of the invention can be isolated using the antigenmicroarray described above.

The term “antibodies” is used herein in a broad sense and includes bothpolyclonal and monoclonal antibodies. In addition to intactimmunoglobulin molecules, also disclosed are antibody fragments orpolymers of those immunoglobulin molecules, and human or humanizedversions of immunoglobulin molecules or fragments thereof, as long asthey are chosen for their ability to interact with the polypeptidesdisclosed herein.

“Antibody fragments” are portions of a complete antibody. A completeantibody refers to an antibody having two complete light chains and twocomplete heavy chains. An antibody fragment lacks all or a portion ofone or more of the chains. Examples of antibody fragments include, butare not limited to, half antibodies and fragments of half antibodies. Ahalf antibody is composed of a single light chain and a single heavychain. Half antibodies and half antibody fragments can be produced byreducing an antibody or antibody fragment having two light chains andtwo heavy chains. Such antibody fragments are referred to as reducedantibodies. Reduced antibodies have exposed and reactive sulfhydrylgroups. These sulfhydryl groups can be used as reactive chemical groupsor coupling of biomolecules to the antibody fragment. A preferred halfantibody fragment is a F(ab). The hinge region of an antibody orantibody fragment is the region where the light chain ends and the heavychain goes on.

Antibody fragments for use in antibody conjugates can bind antigens.Preferably, the antibody fragment is specific for an antigen. Anantibody or antibody fragment is specific for an antigen if it bindswith significantly greater affinity to one epitope than to otherepitopes. The antigen can be any molecule, compound, composition, orportion thereof to which an antibody fragment can bind. An analyte canbe any molecule, compound or composition of interest. For example, theantigen can be a polynucleotide of the invention.

The antibodies or antibody fragments can be tested for their desiredactivity using the in vitro assays described herein, or by analogousmethods, after which their in vivo therapeutic or prophylacticactivities are tested according to known clinical testing methods.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a substantially homogeneous population of antibodies,i.e., the individual antibodies within the population are identicalexcept for possible naturally occurring mutations that may be present ina small subset of the antibody molecules. Also disclosed are “chimeric”antibodies in which a portion of the heavy or light chain is identicalwith or homologous to corresponding sequences in antibodies derived froma particular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is identical with orhomologous to corresponding sequences in antibodies derived from anotherspecies or belonging to another antibody class or subclass, as well asfragments of such antibodies, as long as they exhibit the desiredantagonistic activity (See, U.S. Pat. No. 4,816,567 and Morrison et al.,Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).

The disclosed monoclonal antibodies can be made using any procedurewhich produces monoclonal antibodies. For example, disclosed monoclonalantibodies can be prepared using hybridoma methods, such as thosedescribed by Kohler and Milstein, Nature, 256:495 (1975). In a hybridomamethod, a mouse or other appropriate host animal is typically immunizedwith an immunizing agent to elicit lymphocytes that produce or arecapable of producing antibodies that will specifically bind to theimmunizing agent. Alternatively, the lymphocytes may be immunized invitro, e.g., using the HIV Env-CD4-co-receptor complexes describedherein.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567 (Cabilly et al.). DNAencoding the disclosed monoclonal antibodies can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). Libraries of antibodies oractive antibody fragments can also be generated and screened using phagedisplay techniques, e.g., as described in U.S. Pat. No. 5,804,440 toBurton et al. and U.S. Pat. No. 6,096,441 to Barbas et al.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, such as an Fv,Fab, Fab′, or other antigen-binding portion of an antibody, can beaccomplished using routine techniques known in the art. For instance,digestion can be performed using papain. Examples of papain digestionare described in WO 94/29348 published Dec. 22, 1994 and U.S. Pat. No.4,342,566 which is hereby incorporated by reference in its entirety forits teaching of papain digestion of antibodies to prepare monovaltentantibodies. Papain digestion of antibodies typically produces twoidentical antigen binding fragments, called Fab fragments, each with asingle antigen binding site, and a residual Fc fragment. Pepsintreatment yields a fragment that has two antigen combining sites and isstill capable of cross-linking antigen.

The fragments, whether attached to other sequences, can also includeinsertions, deletions, substitutions, or other selected modifications ofparticular regions or specific amino acids residues, provided theactivity of the antibody or antibody fragment is not significantlyaltered or impaired compared to the non-modified antibody or antibodyfragment. These modifications can provide for some additional property,such as to remove/add amino acids capable of disulfide bonding, toincrease its bio-longevity, to alter its secretory characteristics, etc.In any case, the antibody or antibody fragment must possess a bioactiveproperty, such as specific binding to its cognate antigen. Functional oractive regions of the antibody or antibody fragment may be identified bymutagenesis of a specific region of the protein, followed by expressionand testing of the expressed polypeptide. Such methods are readilyapparent to a skilled practitioner in the art and can includesite-specific mutagenesis of the nucleic acid encoding the antibody orantibody fragment. (Zoller, M. J. Curr. Opin. Biotechnol. 3:348-354,1992).

As used herein, the term “antibody” or “antibodies” can also refer to ahuman antibody or a humanized antibody. Many non-human antibodies (e.g.,those derived from mice, rats, or rabbits) are naturally antigenic inhumans, and thus can give rise to undesirable immune responses whenadministered to humans. Therefore, the use of human or humanizedantibodies in the methods serves to lessen the chance that an antibodyadministered to a human will evoke an undesirable immune response.

The disclosed human antibodies can be prepared using any technique.Examples of techniques for human monoclonal antibody production includethose described by Cole et al. (Monoclonal Antibodies and CancerTherapy, Alan R. Liss, p. 77, 1985) and by Boerner et al. (J. Immunol.,147(1):86-95, 1991). Human antibodies (and fragments thereof) can alsobe produced using phage display libraries (Hoogenboom et al., J. Mol.Biol., 227:381, 1991; Marks et al., J. Mol. Biol., 222:581, 1991).

The disclosed human antibodies can also be obtained from transgenicanimals. For example, transgenic, mutant mice that are capable ofproducing a full repertoire of human antibodies, in response toimmunization, have been described (see, e.g., Jakobovits et al., Proc.Natl. Acad. Sci. USA, 90:2551-255 (1993); Jakobovits et al., Nature,362:255-258 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993)).Specifically, the homozygous deletion of the antibody heavy chainjoining region (J(H)) gene in these chimeric and germ-line mutant miceresults in complete inhibition of endogenous antibody production, andthe successful transfer of the human germ-line antibody gene array intosuch germ-line mutant mice results in the production of human antibodiesupon antigen challenge. Antibodies having the desired activity areselected using Env-CD4-co-receptor complexes as described herein.

Optionally, the disclosed human antibodies can be made from memory Bcells using a method for Epstein-Barr virus transformation of human Bcells. (See, e.g., Triaggiai et al., An efficient method to make humanmonoclonal antibodies from memory B cells: potent neutralization of SARScoronavirus, Nat Med. 2004 August; 10(8):871-5. (2004)), which is hereinincorporated by reference in its entirety for its teaching of a methodto make human monoclonal antibodies from memory B cells). In short,memory B cells from a subject who has survived a natural infection areisolated and immortalized with EBV in the presence of irradiatedmononuclear cells and a CpG oligonuleotide that acts as a polyclonalactivator of memory B cells. The memory B cells are cultured andanalyzed for the presence of specific antibodies. EBV-B cells from theculture producing the antibodies of the desired specificity are thencloned by limiting dilution in the presence of irradiated mononuclearcells, with the addition of CpG 2006 to increase cloning efficiency, andcultured. After culture of the EBV-B cells, monoclonal antibodies can beisolated. Such a method offers (1) antibodies that are produced byimmortalization of memory B lymphocytes which are stable over a lifetimeand can easily be isolated from peripheral blood and (2) the antibodiesisolated from a primed natural host who has survived a naturalinfection, thus eliminating the need for immunization of experimentalanimals, which may show different susceptibility and, therefore,different immune responses.

Antibody humanization techniques generally involve the use ofrecombinant DNA technology to manipulate the DNA sequence encoding oneor more polypeptide chains of an antibody molecule. Accordingly, ahumanized form of a non-human antibody (or a fragment thereof) is achimeric antibody or antibody chain (or a fragment thereof, such as anFv, Fab, Fab′, or other antigen-binding portion of an antibody) whichcontains a portion of an antigen binding site from a non-human (donor)antibody integrated into the framework of a human (recipient) antibody.

To generate a humanized antibody, residues from one or morecomplementarity determining regions (CDRs) of a recipient (human)antibody molecule are replaced by residues from one or more CDRs of adonor (non-human) antibody molecule that is known to have desiredantigen binding characteristics (e.g., a certain level of specificityand affinity for the target antigen). In some instances, Fv framework(FR) residues of the human antibody are replaced by correspondingnon-human residues. Humanized antibodies may also contain residues whichare found neither in the recipient antibody nor in the imported CDR orframework sequences. Generally, a humanized antibody has one or moreamino acid residues introduced into it from a source which is non-human.In practice, humanized antibodies are typically human antibodies inwhich some CDR residues and possibly some FR residues are substituted byresidues from analogous sites in rodent antibodies. Humanized antibodiesgenerally contain at least a portion of an antibody constant region(Fc), typically that of a human antibody (Jones et al., Nature,321:522-525 (1986), Reichmann et al., Nature, 332:323-327 (1988), andPresta, Curr. Opin. Struct. Biol., 2:593-596 (1992)).

Methods for humanizing non-human antibodies are well known in the art.For example, humanized antibodies can be generated according to themethods of Winter and co-workers (Jones et al., Nature, 321:522-525(1986), Riechmann et al., Nature, 332:323-327 (1988), Verhoeyen et al.,Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDRsequences for the corresponding sequences of a human antibody. Methodsthat can be used to produce humanized antibodies are also described inU.S. Pat. No. 4,816,567 (Cabilly et al.), U.S. Pat. No. 5,565,332(Hoogenboom et al.), U.S. Pat. No. 5,721,367 (Kay et al.), U.S. Pat. No.5,837,243 (Deo et al.), U.S. Pat. No. 5,939,598 (Kucherlapati et al.),U.S. Pat. No. 6,130,364 (Jakobovits et al.), and U.S. Pat. No. 6,180,377(Morgan et al.).

The antibodies disclosed herein can also be administered to a subject.Nucleic acid approaches for antibody delivery also exist. The broadlyneutralizing antibodies to the polypeptides disclosed herein andantibody fragments can also be administered to subjects or subjects as anucleic acid preparation (e.g., DNA or RNA) that encodes the antibody orantibody fragment, such that the subject's own cells take up the nucleicacid and produce and secrete the encoded antibody or antibody fragment.

The disclosed compositions and methods can also be used for example astools to isolate and test new drug candidates for a variety of cancers,including, but not limited to lung cancer.

In addition, the compositions described herein may be used as markersfor presence or progression of cancers. The methods and assays describedelsewhere herein may be performed over time, and the change in the levelof reactive polypeptide(s) or polynucleotide(s) evaluated. For example,the assays may be performed every 24-72 hours for a period of 6 monthsto 1 year, and thereafter performed as needed. For example,immunoreactivity to a given polypeptide in individuals with cancer cancorrelate with or predict the development of complications, more severeactivity of disease.

As noted herein, to improve sensitivity, multiple mutations may beassayed within a given sample. Binding agents specific for differentproteins, antibodies, nucleic acids thereto provided herein may becombined within a single assay. Further, multiple primers or probes maybe used concurrently. The selection of c-CBL proteins or genes may bebased on routine experiments to determine combinations that results inoptimal sensitivity. In addition, or alternatively, assays for mutatedc-CBL, antibodies, or nucleic acids specific thereto, provided hereinmay be combined with assays for other known cancer markers or othergenetic markers in subjects with cancer. To assist with such assays,specific biomarkers can assist in the specificity of such tests. Assuch, disclosed herein is a cancer biomarker, wherein the biomarkercomprises c-CBL comprising a mutation that results in an amino acidchange at position S80N, H94Y, Q249E, V391I, 72515_72517delATG, W802*,R830K, A848T, L620F, P170L, S171S, L281F, L254S, or P782L. Alsodisclosed is a cancer biomarker, wherein the biomarker comprises c-CBLcomprising a mutation in a nucleic acid sequence encoding human c-CBL,wherein the mutation is located in the TKB domain, RING finger domain,proline-rich region, C-terminal region, or other domain linkage regionsof c-CBL.

The biomarkers described herein can be in any form that providesinformation regarding presence or absence of a mutation of theinvention. For example, the disclosed biomarkers can be, but is notlimited to a nucleic acid molecule, a polypeptide, or an antibody.

Also disclosed are cancer imaging agents, wherein the agent specificallybinds c-CBL comprising a mutation, wherein the agent binds a c-CBLpolypeptide comprising a mutation at position S80N, H94Y, Q249E, V391I,72515_72517delATG, W802*, R830K, A848T, L620F, P170L, S171S, L281F,L254S, or P782L72515_72517delATG.

The disclosed compositions and methods can be used for targeted genedisruption and modification in any animal that can undergo these events.Gene modification and gene disruption refer to the methods, techniques,and compositions that surround the selective removal or alteration of agene or stretch of chromosome in an animal, such as a mammal, in a waythat propagates the modification through the germ line of the mammal. Ingeneral, a cell is transformed with a vector which is designed tohomologously recombine with a region of a particular chromosomecontained within the cell, as for example, described herein. Thishomologous recombination event can produce a chromosome which hasexogenous DNA introduced, for example in frame, with the surroundingDNA. This type of protocol allows for very specific mutations, such aspoint mutations, to be introduced into the genome contained within thecell. Methods for performing this type of homologous recombination aredisclosed herein.

One of the preferred characteristics of performing homologousrecombination in mammalian cells is that the cells should be able to becultured, because the desired recombination events occur at a lowfrequency.

Once a cell is produced through the methods described herein, an animalcan be produced from this cell through either stem cell technology orcloning technology. For example, if the cell into which the nucleic acidwas transfected was a stem cell for the organism, then this cell, aftertransfection and culturing, can be used to produce an organism whichwill contain the gene modification or disruption in germ line cells,which can then in turn be used to produce another animal that possessesthe gene modification or disruption in all of its cells. In othermethods for production of an animal containing the gene modification ordisruption in all of its cells, cloning technologies can be used. Thesetechnologies generally take the nucleus of the transfected cell andeither through fusion or replacement fuse the transfected nucleus withan oocyte which can then be manipulated to produce an animal. Theadvantage of procedures that use cloning instead of ES technology isthat cells other than ES cells can be transfected. For example, afibroblast cell, which is very easy to culture can be used as the cellwhich is transfected and has a gene modification or disruption eventtake place, and then cells derived from this cell can be used to clone awhole animal.

Disclosed herein are transgenic animals comprising mutations in anucleotide sequence capable of encoding a c-CBL protein. Transgenicanimals include, but are not limited to zebrafish and nematodes. It isalso understood that the animal can comprise any mammal. For example,the animal can be a mouse, vole, rat, guinea pig, cat, dog, cow, sheeppig, monkey, or human. For example, disclosed are transgenic animalcomprising one or more of the disclosed c-CBL mutations including, butnot limited to c-CBL encoding nucleic acids comprising a mutation at anucleic acid position corresponding to a change in amino acid atposition S80N, H94Y, Q249E, V391I, 72515_72517delATG, W802*, R830K,A848T, L620F, P170L, S171S, L281F, L254S, or P782L. Also disclosed aretransgenic animals comprising one or more of the disclosed c-CBLmutations including, but not limited to c-CBL encoding nucleic acidsthat comprises a mutation in a nucleic acid sequence encoding humanc-CBL, wherein the mutation is located in the TKB domain, RING fingerdomain, proline-rich region, or the C-terminal region of c-CBL.

It is understood that the disclosed nucleic acids and proteins can berepresented as a sequence consisting of the nucleotides of amino acids.There are a variety of ways to display these sequences, for example thenucleotide guanosine can be represented by G or g. Likewise the aminoacid valine can be represented by Val or V. Those of skill in the artunderstand how to display and express any nucleic acid or proteinsequence in any of the variety of ways that exist, each of which isconsidered herein disclosed. Specifically contemplated herein is thedisplay of these sequences on computer readable mediums, such as,commercially available floppy disks, tapes, chips, hard drives, compactdisks, and video disks, or other computer readable mediums. Alsodisclosed are the binary code representations of the disclosedsequences. Those of skill in the art understand what computer readablemediums. Thus, computer readable mediums on which the nucleic acids orprotein sequences are recorded, stored, or saved.

Disclosed are computer readable mediums comprising the sequences andinformation regarding the sequences set forth herein. Also disclosed arecomputer readable mediums comprising the sequences and informationregarding the sequences set forth herein.

Also disclosed herein is a computer-readable medium comprising humanc-CBL amino acid polypeptide sequence comprising a mutation at positionS80N, H94Y, Q249E, V391I, 72515_72517delATG, W802*, R830K, A848T, L620F,P170L, S171S, L281F, L254S, or P782L and/or nucleic acid sequenceencoding a human c-CBL polypeptide comprising a mutation at a nucleicacid position corresponding to a change in amino acid at position S80N,H94Y, Q249E, V391I, 72515_72517delATG, W802*, R830K, A848T, L620F,P170L, S171S, L281F, L254S, or P782L. Further disclosed is acomputer-readable medium comprising human c-CBL amino acid polypeptidesequence comprising a mutation in a nucleic acid sequence encoding humanc-CBL, wherein the sequence comprises a mutation in a nucleic acidsequence encoding human c-CBL, wherein the mutation is located in theTKB domain, RING finger domain, proline-rich region, C-terminal region,or other domain linkage regions of c-CBL.

The computer-readable mediums disclosed herein can comprise a storagemedium for sequence information for one or more subjects. For example,the information can be a personalized genomic profile for a subjectknown or suspected to have a cancer, wherein the genomic profilecomprises sequence information for c-CBL comprising one or more of themutations disclosed herein.

The present invention therefore also provides predictive, diagnostic,and prognostic kits comprising degenerate primers to amplify a targetnucleic acid in the c-CBL gene and instructions comprising amplificationprotocol and analysis of the results. The kit may alternatively alsocomprise buffers, enzymes, and containers for performing theamplification and analysis of the amplification products. The kit mayalso be a component of a screening, diagnostic or prognostic kitcomprising other tools such as DNA microarrays. Preferably, the kit alsoprovides one or more control templates, such as nucleic acids isolatedfrom normal tissue sample, and/or a series of samples representingdifferent variances in the c-CBL gene.

In one embodiment, the kit provides two or more primer pairs, each paircapable of amplifying a different region of the c-CBL gene (each regiona site of potential variance) thereby providing a kit for analysis ofexpression of several gene variances in a biological sample in onereaction or several parallel reactions. Primers in the kits may belabeled, for example fluorescently labeled, to facilitate detection ofthe amplification products and consequent analysis of the nucleic acidvariances. For example, the primers can be one or more of the primers ofTable 3.

In one embodiment, more than one variance can be detected in oneanalysis. A combination kit will therefore comprise of primers capableof amplifying different segments of the kinase domain of the c-CBL gene.For example, the primers can be one or more of the primers of Table 3.The primers may be differentially labeled, for example using differentfluorescent labels, so as to differentiate between the variances. Theprimers contained within the kit may include primers selected fromcomplementary sequences to the coding sequence of c-CBL. For example,the primers can be one or more of the primers of Table 1.

In further embodiments, the invention provides immunological kits foruse in detecting the activation levels of downstream targets of c-CBL'sE3 ubiquitination activity. The kit generally comprises, a) apharmaceutically acceptable carrier; b) an c-MET and Ubiquitin antibodydirected against a downstream targets of c-CBL's E3 ubiquitinationactivity; and c) an immunodetection reagent. Antibodies (monoclonal orpolyclonal) are commercially available and may also be prepared bymethods known to those of skill in the art, for example, in CurrentProtocols in Immunology, John Wiley & Sons, Edited by: John E. Coligan,Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober,2001.

The immunodetection kits of the invention may additionally contain oneor more of a variety of other cancer marker antibodies or antigens, ifso desired. Such kits could thus provide a panel of cancer markers, asmay be better used in testing a variety of patients. By way of example,such additional markers could include, other tumor markers such as PSA,SeLe (X), HCG, as well as p53, cyclin D1, p16, tyrosinase, MAGE, BAGE,PAGE, MUC18, CEA, p2′7, [bgr]HCG or other markers known to those ofskill in the art.

The container means of the kits will generally include at least onevial, test tube, flask, bottle, or even syringe or other containermeans, into which the antibody or antigen may be placed, and preferably,suitably aliquoted. Where a second or third binding ligand or additionalcomponent is provided, the kit will also generally contain a second,third or other additional container into which this ligand or componentmay be placed. The kits of the present invention will also typicallyinclude a means for containing the antibody, antigen, and any otherreagent containers in close confinement for commercial sale. Suchcontainers may include injection or blow-molded plastic containers intowhich the desired vials are retained.

Methods

The disclosed compositions, including the c-CBL mutations disclosedherein can be used in a variety of different methods, for example inprognostic, predictive, diagnostic, and therapeutic methods and as avariety of different compositions. For example, disclosed herein aremethods of identifying a subject that is susceptible to treatment with ac-Met inhibitor comprising determining whether a sample from the subjectcomprises a mutation in a nucleic acid sequence encoding human c-CBL.

Due to the vast information supporting the role of c-Met and HGF in thepathogenesis of human cancers along with successes of other RTKinhibitors, a number of approaches have been attempted to inhibit HGF-or c-Met-dependent signaling. These approaches include: (1) c-Metbiologic inhibitors (ribozymes, dominant-negative receptors, decoyreceptors, peptides); (2) HGF kringle variant antagonists; (3) HGFantagonist antibodies; (4) c-Met antagonist antibodies; and (5)small-molecule c-Met inhibitors. To date, several possible c-Metinhibitors have been developed with the intent on either silencing, ordecreasing c-Met expression or decreasing c-Met activity. For example,Compound X, PHA665752 (Pfizer, Inc.), SU11274 (Sugen, Inc.), SU11271(Sugen, Inc.), SU11606 (Sugen, Inc.), ARQ197 (ArQuleArqule, Inc.), MP470(Supergen, Inc.), Kirin, XL-880 (Exelixis, Inc.), XL184 (Exelixis, Inc.)Geldanamycins, SGX523 (SGX, Inc.), MGCD265 (MethylGene, Inc.), HPK-56(Supergen, Inc.), AMG102 (Amgen, Inc.), MetMAb (Genentech, Inc.),ANG-797 (Angion Biomedica Corp.), CGEN-241 (Compugen LTD.), Metro-F-1(Dompe S.p.A.), ABT-869 (Abbott Laboratories) and K252a are all c-Metinhibitors currently being produced. In addition, h224G11 (Abbott),ARQ197 (ArQule), AMG208 (Amgen), BMS907351 (Bristol-Myers SquibbCompany), DP3590 (Deciphera Pharmaceuticals), DP4157 (DecipheraPharmaceuticals, LLC), E7050 (Eisai Co.), SGX523 (Eli Lilly), XL880(Exelixis), XL184 (Exelixis), RG3638 (MetMab), GSK1363089(GlaxoSmithKline), INCB28060 (Incyte), MK2461 (Merck & Co), EMD1204831(Merck KGaA), EMD1214063 (Merck Serono S.A.), MGCD265 (MethylGene),PF2341066 (Pfizer), MP470 (SuperGen), AMG102 (Amgen), ABT-869 (AbbottLaboratories), ANG-797 (Angion Biomedica Corp), CGEN-241 (Compugen LTD),PHA665752 (Pfizer), SU11274 (Sugen), SU11271 (Sugen), SU11606 (Sugen),HPK-56 (Supergen), K252a (Merck) are also c-Met inhibitors currentlybeing produced.

Cancers or cancer tissues that can be used in the disclosed methodsinclude, but are not limited to, lymphoma (Hodgkins and non-Hodgkins)B-cell lymphoma, T-cell lymphoma, leukemia such as myeloid leukemia andother types of leukemia, mycosis fungoide, carcinoma, adenocarcinoma,sarcoma, glioma, astrocytoma, blastoma, neuroblastoma, plasmacytoma,histiocytoma, melanoma, adenoma, hypoxic tumour, myeloma, AIDS-relatedlymphoma or AIDS-related sarcoma, metastatic cancer, bladder cancer,brain cancer, nervous system cancer, squamous cell carcinoma of the headand neck, neuroblastoma, glioblastoma, ovarian cancer, skin cancer,liver cancer, squamous cell carcinomas of the mouth, throat, larynx, andlung, colon cancer, cervical cancer, breast cancer, cervical carcinoma,epithelial cancer, renal cancer, genitourinary cancer, pulmonary cancer,esophageal carcinoma, lung cancer, head and neck carcinoma,hematopoietic cancer, testicular cancer, colo-rectal cancer, prostaticcancer, and pancreatic cancer. Specific lung cancers that can be used inthe disclosed methods include, but are not limited to Non-Small CellLung Cancers (NSCLC) and/or Small Cell Carcinomas (SCC).

While some candidates appear to be successful in inhibiting wild-typec-Met in vitro, it is unknown how c-met inhibitors will affect asubject.

Disclosed herein are methods of identifying a subject that issusceptible to treatment with a c-Met inhibitor comprising determiningwhether a sample from the subject comprises a mutation in a nucleic acidsequence encoding human c-CBL, wherein the mutation results in an aminoacid change at position S80N, H94Y, Q249E, V391I, 72515_72517delATG,W802*, R830K, A848T, L620F, P170L, S171S, L281F, L254S, or P782L. Thepresence of a mutation in a nucleic acid sequence encoding human c-CBLcan then identify a subject that is susceptible to treatment with ac-Met inhibitor.

Also disclosed are methods of identifying a subject that is susceptibleto treatment with a c-Met inhibitor comprising determining whether asample from the subject comprises a mutation in a nucleic acid sequenceencoding human c-CBL, wherein the mutation is located in the TKB domain,RING finger domain, proline-rich region, C-terminal region, or otherdomain linkage regions of c-CBL. For example, the absence of a mutationin a nucleic acid sequence encoding human c-CBL identifies a subjectthat is not susceptible to treatment with a c-Met inhibitor. In someaspects, the presence of a mutation in a nucleic acid sequence encodinghuman c-CBL identifies a subject that is susceptible to treatment with ac-Met inhibitor. The mutation located in the TKB domain, RING fingerdomain, proline-rich region, C-terminal region, or other domain linkageregions of c-CBL can be, but is not limited to a mutation results in anamino acid change at position S80N, H94Y, Q249E, V391I,72515_72517delATG, W802*, R830K, A848T, L620F, P170L, S171S, L281F,L254S, or P782L.

Also disclosed are methods of identifying a cancer that is susceptibleto treatment with a c-Met inhibitor, comprising determining whether asample from the cancer comprises a mutation in a nucleic acid sequenceencoding human c-CBL. For example, in some aspects, the absence of amutation in a nucleic acid sequence encoding human c-CBL can identify asubject that is not susceptible to treatment with a c-Met inhibitor. Inother aspects, the presence of a mutation in a nucleic acid sequenceencoding human c-CBL can a subject that is susceptible to treatment witha c-Met inhibitor.

Also disclosed are methods of determining responsiveness of a cancer ina subject to treatment with a c-Met inhibitor, said method comprisingdetermining whether a cancer sample from a subject who has been treatedwith the c-Met inhibitor comprises a mutation in a nucleic acid sequenceencoding human c-CBL, wherein the mutation results in an amino acidchange at position S80N, H94Y, Q249E, V391I, 72515_72517delATG, W802*,R830K, A848T, L620F, P170L, S171S, L281F, L254S, or P782L, whereinpresence of the mutated nucleic acid sequence is indicative that thecancer is responsive to treatment with the c-Met inhibitor.

Also disclosed are methods of determining responsiveness of a cancer ina subject to treatment with a c-Met inhibitor, said method comprisingdetermining whether a cancer sample comprises a mutation in a nucleicacid sequence encoding human c-CBL, wherein the mutation is located inthe TKB domain, RING finger domain, proline-rich region, C-terminalregion, or other domain linkage regions of c-CBL, wherein presence ofthe mutated nucleic acid sequence is indicative that the cancer isresponsive to treatment with the c-Met inhibitor.

Also disclosed are methods of detecting cancer in a sample comprisingdetermining whether the sample comprises a mutation in a nucleic acidsequence encoding human c-CBL, wherein the mutation results in an aminoacid change at position S80N, H94Y, Q249E, V391I, 72515_72517delATG,W802*, R830K, A848T, L620F, P170L, S171S, L281F, L254S, or P782L.

Also disclosed are methods of detecting cancer in a sample comprisingdetermining whether the sample comprises a mutation in a nucleic acidsequence encoding human c-CBL, wherein the mutation is located in theTKB domain, RING finger domain, proline-rich region, C-terminal region,or other domain linkage regions of the nucleic acid sequence encodinghuman c-CBL.

Susceptibility can either mean that the cancer sample comprising amutation in a nucleic acid sequence encoding human c-CBL, wherein themutation results in an amino acid change at position S80N, H94Y, Q249E,V391I, 72515_72517delATG, W802*, R830K, A848T, L620F, P170L, S171S,L281F, L254S, or P782L, is less responsive to a c-Met inhibitor or moreresponsive to a c-Met inhibitor.

Also disclosed are methods of identifying a subject that is susceptibleto treatment with a c-Met inhibitor comprising determining the level ofc-CBL expression in a sample from the subject. For example, disclosedare methods of identifying a subject that is susceptible to treatmentwith a c-Met inhibitor comprising determining the level of c-CBLexpression in a sample from the subject, wherein low levels of c-CBLexpression identify a subject that is susceptible to treatment with ac-Met inhibitor. “low levels” of c-CBL expression can mean levels thatare below normal levels of c-CBL expression.

Also disclosed are methods of identifying a subject that is susceptibleto treatment with a c-Met inhibitor comprising determining the level ofc-CBL expression in a sample from the subject, wherein high levels ofc-CBL expression identify a subject that is not susceptible to treatmentwith a c-Met inhibitor. “High levels” of c-CBL expression can meanlevels that are equal or above normal levels of c-CBL expression.

Also disclosed are methods of determining responsiveness of a cancer ina subject to treatment with a c-Met inhibitor, said method comprisingdetermining the expression level of c-CBL in a cancer sample from thesubject, wherein equal or higher levels of c-CBL expression in thecancer sample relative to a normal sample are indicative that the canceris not susceptible to treatment with the c-Met inhibitor.

Also disclosed are methods of determining responsiveness of a cancer ina subject to treatment with a c-Met inhibitor, said method comprisingdetermining the expression level of c-CBL in a cancer sample from thesubject, wherein lower levels of c-CBL expression in the cancer samplerelative to a normal sample are indicative that the cancer issusceptible to treatment with the c-Met inhibitor.

In the methods described herein, the expression level of c-CBL can bedetermined by determining the amount of c-CBL RNA or c-CBL protein inthe sample. Methods for determining the amount of RNA or protein presentin a sample are well known in the art. Any known methods for suchdeterminations can be used in the methods described herein. For example,protein levels can be determined by methods including, but not limitedto, western blotting or immunohistochemistry. Also, RNA levels can bedetermined by methods including, but not limited to, RT-PCR, quantitiveRT-PCR, expression array analysis, RNase protection assay or northernblotting.

In the methods described herein the expression level of c-CBL can alsobe determined by determining the presence of loss of heterozygocity(LOH) at the c-CBL gene locus, wherein LOH at the c-CBL locus isindicative of lower c-CBL expression.

The transgenic animals described above can also be used in any of themethods described herein. For example, the transgenic animals describedherein can be used to identify a cancer that is or is not susceptible totreatment with a c-met inhibitor wherein the transgenic animal comprisesa mutation in a nucleic acid sequence encoding human c-CBL, wherein themutation results in an amino acid change at position S80N, H94Y, Q249E,V391I, 72515_72517delATG, W802*, R830K, A848T, L620F, P170L, S171S,L281F, L254S, or P782L.

The transgenic animals described herein can also be used to identify acancer that is susceptible or not susceptible to treatment with a c-metinhibitor, wherein the transgenic animal comprises a mutation in anucleic acid sequence encoding human c-met, wherein the sequence ismutated in a nucleic acid sequence encoding human c-CBL, wherein themutation is located in the TKB domain, RING finger domain, proline-richregion, or the C-terminal region of c-CBL.

The transgenic animals described herein can also be used to determinethe responsiveness of a cancer in a subject to treatment with a c-metinhibitor, wherein the transgenic animal comprises a mutation in anucleic acid sequence encoding human c-CBL, wherein the mutation resultsin an amino acid change at position S80N, H94Y, Q249E, V391I,72515_72517delATG, W802*, R830K, A848T, L620F, P170L, S171S, L281F,L254S, or P782L, wherein presence of the mutated nucleic acid sequenceis indicative that the cancer is responsive to treatment with the c-metinhibitor.

The invention will be further described with reference to the followingexamples; however, it is to be understood that the invention is notlimited to such examples. Rather, in view of the present disclosure thatdescribes the current best mode for practicing the invention, manymodifications and variations would present themselves to those of skillin the art without departing from the scope and spirit of thisinvention. All changes, modifications, and variations coming within themeaning and range of equivalency of the claims are to be consideredwithin their scope.

EXAMPLES Example 1 Tissue Samples

Paired tumor and normal lung tissues were obtained from 30 NSCLCpatients who were recruited at the Taipei Veterans General Hospitalafter obtaining appropriate institutional review board permission andinformed consent from the patients. Among them, 28 were men and 2 werewomen, with aged at diagnosis ranging from 47 to 90 years. For tumortypes, 14 were adenocarcinoma, 10 were squamous cell carcinoma, 5 werelarge cell carcinoma, and 1 was adenosquamous carcinoma. For tumorstage, 13 were stage I, 4 were stage II, 9 were stage III, and 4 werestage IV.

Cell Culture

Human non-small cell lung carcinoma cell A549 was maintained in DMEM.Media was supplemented with 10% fetal bovine serum, 100 units/ml ofpenicillin, and 100 g/ml of streptomycin (Invitrogen, Eugene, Oreg.).The cell was maintained at 37° C. in a humidified incubator containing5% CO2 in air.

A549 and H226 cells are generally accepted predictive cell models forcarcinoma behavior, for example, NSCLC and head and neck carcinoma.(Pasqualetti et al., Lung Cancer, 2011; Shi et al., Oncogene, 2011)

Analysis of c-CBL Gene Mutation

Exons 2 to 16 of c-CBL gene were individually amplified by polymerasechain reaction (PCR). Primers are listed in Table 3. PCR conditions wereone cycle of 95° C. 5 minutes, 35 cycles of 94° C. 30 seconds, 58° C. 30seconds, 72° C. 2 minutes, and one cycle of 72° C. 10 minutes. PCRproducts were treated with ExoSAP-IT (USB Corp., Cleveland, Ohio, USA)and sequenced by Big Dye Terminator Chemistry (Applied Biosystems,Weiterstadt, Germany). Sequencing was performed on the forward codingstrand with confirmation of c-CBL alterations performed by sequencingthe reverse strand as well. Chromatograms were analyzed for mutationsusing Mutation Surveyor v2.61 (Softgenetics).

Plasmid Constructs and Site-Directed Mutagenesis

The wild-type c-CBL cDNA insert was subcloned into the expression vectorpCDNA3.1_N-mCherry, using BamHI and XhoI restriction enzyme sites. Usingthe parental plasmid pcDNA3.1-NmCherry containing the subclonedfull-length wild-type c-CBL cDNA insert, TKB domain point mutation(Q249E) of c-CBL was created using the (SEQ ID NO:31)5′-CTTTACCCGACTCTTTGAGCCCTGGTCCTCTTTGC-3′ primer and its complementaryprimer with QuickChange Site-Directed Mutagenesis XL kit (Stratagene, LaJolla, Calif.) according to the manufacturer's instructions. The pointmutation constructs were confirmed by standard DNA sequencing of bothstrands.

TABLE 3 SEQ ID SEQ ID Exon NO Forward (5′ to 3′) NO Reverse (5′ to 3′) 2 SEQ ID TAAAATGGTTGCCTGTGGGCAATG SEQ ID TGTGTTACCCATTCAGGCACTC NO: 3NO: 15  3 SEQ ID CATCTTGTATGGTGAATTTGGTGC SEQ IDGACTCCGTCTCAAAAAGAAACCAC NO: 4 NO: 16  4 SEQ ID GCTTAATGTGGCTCTCCTTCCSEQ ID GTGAGGAGAAGAAAGCAGTTGG NO: 5 NO: 17  5 SEQ IDCTCTGAGTTGGTTGTACATCTGAC SEQ ID CAGAACCTTGGCTATTGCGAAAC NO: 6 NO: 18  6SEQ ID GTCTGTATCTTGCCTTGCCTTC SEQ ID CCTAAGTTCCCAGACTCTAACAGATG NO: 7NO: 19  7 & 8 SEQ ID GGACCCAGACTAGATGCTTTCTG SEQ IDCCTTGTATCAGTAAAGGCTATATAAT NO: 8 NO: 20 ACC  9 SEQ IDCGGTATTATATAGCCTTTACTGATACAAGG SEQ ID CCAGTCTCCTAAACTGCCATCTTAC NO: 9NO: 21 10 & 11 SEQ ID CCTAGGTCTGGCCCATTTGTAG SEQ IDCTGGCCCACACATATTTCTTAACAG NO: 10 NO: 22 12 SEQ ID CAGAGGCTCAGCTGTGGTAAGSEQ ID CAGAGCAATGAAACAGATGGCAG NO: 11 NO: 23 13 & 14 SEQ IDGCTCTGTTCAATTTGAGTTATGTCTG SEQ ID GCTTAGATCAAGCTATCTCAATTGCC NO: 12NO: 24 15 SEQ ID GTTGGCCCACAGTAGACAATC SEQ ID CTTGGGACTTTCCTCCCATTTAGACNO: 13 NO: 25 16 SEQ ID CTTGTGACTGAAGAGCACATGTAC SEQ IDCTAGGTGCCACTTGAGTAATAACTC NO: 14 NO: 26

Table 3 provides a listing of c-CBL amplification primers used in anexperimental example described herein.

Transfection of c-CBL Constructs

A549 cell line was transfected using the ExGen500 (Fermentas, GlenBurnie, Md.) reagents according to the manufacturer's instructions. Twoμg of expression plasmid DNA, containing either no insert (emptyvector), wild-type c-CBL, or Q249E c-CBL, was used for transfection foreach 6 well culture plate.

Cell Viability Assay

Cells were transfected with pcDNA 3.1_N-mCherry empty vector, c-CBL wildtype, and c-CBL mutant Q249E constructs. After 48 h transfection, cellsstained with Trypan Blue solution (0.4%) (Sigma-Aldrich, St. Louis, Mo.)was used to measure the effect of c-CBL mutation on cell growth.

A Group of Taiwanese Lung Cancer Patients Carried Mutations in c-CBLGene.

Recent reports have indicated several c-CBL mutations in AML. (Reindl C,et al. Clin Cancer Res 15:2238-2247 (2009); Caligiuri M A, et al., Blood110:1022-1024 (2007)). A genomic DNA sequence screening of paired tumorand normal samples from 30 Taiwanese lung cancer patients was performedto investigate the role of c-CBL in lung cancer. Twelve (12) pairs ofprimers were prepared to sequence the coding region of c-CBL gene fromexons 2 to 16 (Table 3). Two exonic mutations in c-CBL were identified.One is a known single nucleotide polymorphism (SNP) L620F in exon 11 ofpatients TW9 and TW18. Importantly, another is a non-synonymous mutationQ249E, which was confirmed by sequencing both strands of c-CBL gene inDNA from tumor tissue of patient TW30 (Tables 4, 5 and FIG. 1A).Moreover, there was no Q249E mutation shown in corresponding normaltissue, suggesting that Q249E is a somatic mutation. The novel somaticmutation Q249E is located in TKB domain and SNP L620F is in proline-richregion (FIG. 1B). In addition, seven mutations were found in intronincluding a known SNP 67960 het_delT in intron 4 and other novel SNPs(Table 4), which were also confirmed by sequencing both strands of c-CBLgene in DNA from tumor and corresponding normal tissue. The totalmutation rate for tumor was 30% (9/30) and for normal was 27% (8/30).The different tumor types showed different mutation rates in theTaiwanese lung cancer population. There were 77.8% (7/9) inadenocarcinoma (AD), 100% (9/9) in squamous cell carcinoma (SQ), 33.3%(3/9) in adenosquamous carcinoma (AD/SQ), and 66.7% (6/9) in large cellcarcinoma (LC) (FIG. 2). However, due to the small number analysis, Themutation rates between AD and SQ (P=0.133), between AD and AD/SQ(P=0.058), and between AD and LC (P=0.599) were not statisticallysignificant.

TABLE 4 Numbers of sample Location Mutation Domain (Frequency) exon 467885 C > CG (Q249E) TKB 1 (3.3%) Intron 4 67960 het_delT* 30 (100%)67968 del C 2 (13.3%) Intron 5 68861_68862 het_insT 19 (63.3%)68861_68862 insT 3 (10%) Intron 8 72287 del T 29 (96.7%) 72315 G > GT 13(43.3%) Intron 9 78771 G > T 20 (66.7%) exon II 79346 C > CT (L620F)**Pro_rich 2 (13.3%) Known SNP: rs 3842642; **Known SNP: rs2227988; TKB:tyrosine kinase binding domain; Pro_rich: proline-rich region.

Table 4 summarizes the analysis of c-CBL mutations that occurred intumor tissues of 30 Taiwanese lung cancer patients during the experimentdescribed in Table 3.

Since c-CBL protein interacts with c-Met or EGFR, mutations in c-Met andEGFR of this cohort of 30 Taiwanese lung cancer patients were analyzed.The mutations in c-Met and EGFR were analyzed in both tumor andcorresponding normal tissues and were previously identified.(Jagadeeswaran R, et al., Cancer Res 68:132-142 (2008)). The dataindicated that patients with c-CBL Q249E somatic mutant showed no c-Metand EGFR mutations, but two patients with c-CBL L620F SNP had eitherN375S SNP in c-Met gene or L858R mutation in EGFR gene (Table 5).

TABLE 5 No. Sex Tumor type XStage c-Cbl c-Met EGFR TW 1 M AD IIIA NF NFDel: 747-751 TW 2 M AD IIA NF NF L858R TW 3 F AD IB NF NF Del: 746-750Ins A TW 4 M AD IIIA NF NF Del: 748-752 TW 5 M AD IB NF NF Del: 747-753Ins S TW 6 M AD IIIA NF N375S Del: 746-750 TW 7 F AD/SQ IIB NF NF Del:746-750 TW 8 M AD IV NF NF Del: 746-750 TW 9 M AD IB L620F NF L858R TW10M AD IV NF NF L858R TW11 M AD IV NF NF Del: 746-750 TW12 M AD IB NFN375S NF TW13 M SQ IB NF NF Del: 746-750 TW14 M AD IB NF L211W NF TW15 MSQ IIB NF N375S NF TW16 M LC IIIA NF NF NF TW17 M SQ IIIA NF N375S* NFTW18 M SQ IIIA L620F N375S NF TW19 M LC IB NF NF NF TW20 M SQ IIIA NF NFL858R TW21 M LC IB NF N375S NF TW22 M AD IB NF N375S NF TW23 M LC IB NFNF NF TW24 M SQ IV NF NF NF TW25 M SQ IIIB NF N375S NF TW26 M SQ IB NFN375S NF TW27 M AD IIIA NF N375S NF TW28 M SQ IB NF N375S NF TW29 M LCIIB NF NF NF TW30 M SQ IB Q249E NF NF AD: adenocarcinoma; SQ: squamouscell carcinoma; LC: large cell carcinoma; NF: not found; *homozygousmutation.

Table 5 summarizes c-CBL, c-Met, and EGFR exon mutations that were foundin Taiwanese lung cancer patients during the experiment described inTables 3 and 4.

c-CBL Mutations Increase the Lung Cancer Cell Viability.

To investigate the effect of c-CBL mutation in cell biologicalfunctions, the c-CBL wild-type and Q249E mutant expression constructswere made into pcDNA3.1-NmCherry vector (FIG. 3), which carried cherryred fluorescent in C-terminal and was used to confirm the transfectionefficiency. Secondly, A549 cell showing low c-CBL basal expression wasused so that the effect of exogenous c-CBL on cell viability wasobserved unambiguously without the complication of endogenous c-CBL.Control empty vector, c-CBL wild-type, or c-CBL Q249E mutant wastransfected to A549 cell line and the cell viability was evaluated at 48h post-transfection. A549 transfected cells showed 102.5% (P=0.868)viability in c-CBL wild-type and 135% (P=0.049) viability in Q249Emutant compared to the empty control vector (FIG. 4).

The results demonstrated that c-CBL mutation occurs in lung cancerpatients. In addition, the novel c-CBL somatic mutation, Q249E, showedan increasing viability compared to wild-type c-CBL in A549 cell model.A recent study showed that there is no somatic mutation andamplification in c-Met but protein overexpression in Taiwanese lungcancer. (Engelman J A, et al., Science 316:1039-1043 (2007)). Inaddition, mutations in EGFR are observed frequently in NSCLC patients inEast Asian populations.

Polyubiquitination of RTKs requires a functional c-CBL TKB domain, whichinteracts with receptor targets. A novel c-CBL somatic mutation Q249E inTKB domain was discovered. It is contemplated that the novel c-CBLsomatic mutation Q249E in TKB domain can lose the ability to interactand degrade its target RTKs. The cell viability results indicated thatQ249E mutant increased cell proliferation, showing the importance of TKBdomain. Hence, it is contemplated that the loss of negative controlthrough c-CBL mutations that delete c-CBL binding site function cancontribute to the deregulation of c-Met or other RTKs observed incancers. The patient with c-CBL Q249E mutation did not show mutation ineither c-Met or EGRF gene. Thus, it is contemplated that c-CBL mutationcan lead to overexpression of c-Met and EGFR proteins without mutationin corresponding genes.

Example 2 Tissue Samples

Lung cancer tissue and paired adjacent normal lung tissues were obtainedfrom 50 Caucasian, 29 African-Americans and 40 Taiwanese NSCLC patientswho were recruited at the University of Chicago Hospital (Chicago, USA)(Caucasian and African-American patients) and Taipei Veterans GeneralHospital of Taiwan (Taiwanese patients). Out of 119 samples, 77 weremen, 38 were women and 4 were unknown with age at diagnosis ranging from47 to 90 years. In terms of tumor types, 53 were adenocarcinoma, 32 weresquamous cell carcinoma and 34 were large cell carcinoma. 49 were stageI, 14 were stage II, 34 were stage III, and 13 were stage IV.

Cell Culture

Human non-small cell lung carcinoma cells A549 and H358 were maintainedin DMEM and RPMI-1640, respectively. Human embryonic kidney 293T cellswere cultured in DMEM. Media were supplemented with 10% fetal bovineserum, 100 units/ml of penicillin, and 100 mg/ml of streptomycin(Invitrogen, Carlsbad, Calif.). Cells were cultured at 37° C. in ahumidified incubator containing 5% CO2.

c-CBL Gene Mutational Analysis

Exons 2 to 16 of c-CBL gene were individually amplified by polymerasechain reaction (PCR). PCR conditions were 1 cycle of 95 uC. for 5minutes; 35 cycles of 94° C. for 30 seconds, 58° C. for 30 seconds and72° C. for 2 minutes; and one cycle of 72° C. for 10 minutes. PCRproducts were treated with ExoSAP-IT (USB Corporation, Cleveland, Ohio)and sequenced by Big-Dye Terminator Chemistry (Applied Biosystems,Foster City, Calif.). Sequencing was performed on the forward codingstrand with confirmation of c-CBL alterations performed by sequencingthe reverse strand as well. Chromatograms were analyzed for mutationsusing Mutation Surveyor v2.61 (Softgenetics, State College, Pa.).

Plasmid Constructs and Site-Directed Mutagenesis

The wild-type c-CBL cDNA insert was subcloned into the pAlterMaxexpression vector using XhoI and SalI restriction enzyme sites (Promega,Madison, Wis.). Using this parental plasmid pAlterMax-c-CBL, the TKBdomain double mutation (S80N/H94Y), the point mutation (Q249E), and theC-terminal point mutation W802* of c-CBL were created using thefollowing primers: SEQ ID NO:275′-GCTGGCGCTAAAGAATAACCCACCTTATATCTTAGAC-3′ and SEQ ID NO:285′-CTACCAGATACCTACCAGTATCTCCGTACTATCTTGTC-3′ for the double mutationS80N/H94Y; SEQ ID NO:29 5′-CTTTACCCGACTCTTTGAGCCCTGGTCCTCTTTGC-3′ forQ249E, and SEQ ID NO:30 5′-AGCTCCTCCTTTGGCTGATTGTCTCTGGATGGTGATC-3′ forW802* along with their complementary primers using the QuickChangeSite-Directed Mutagenesis XL kit (Stratagene, La Jolla, Calif.)according to the manufacturer's instructions. The constructs wereconfirmed for the point mutations by standard DNA sequencing of bothstrands.

Loss of Heterozygosity (LOH) Analysis

Five microsatellites on chromosome 11 (3 on 11q at or within 200 kb upor downstream of the c-CBL gene and 2 control markers on 11p) wereselected for analysis. Established microsatellite markers and respectiveprimer sequences were selected from the GeneLoc database(http://genecards.weizmann.ac.il/geneloc/index.shtml, Weizmann Instituteof Science, Rehovot, Israel). Primers were custom designed and eachforward primer was fluorescently labeled at the 59 end with FAM, PET,NED, or VIC (Applied Biosystems). Primer annealing temperatures andduplex scores were evaluated with NIST Primer Tools(http://yellow.nist.gov:8444/dnaAnalysis/primerToolsPage.do; NationalInstitute of Standards and Technology, Gaithersburg, Md.). Primers wereverified by performing PCR with control DNA (isolated from TK6 cells)and resolving the products on agarose gels. Bands were visualized withan UV transilluminator. Genomic DNA was extracted from tumor samples andpaired normal lung tissue. Primers were grouped into multiplexcombinations. Marker D11S929 served as an internal control to check forconsistency in PCRs and of peaks from capillary electrophoresis.Multiplex PCRs were carried out in a volume of 10 mL that contained 1 mLgenomic DNA (20-50 ng), 0.5 mM of each primer (1.0 mM total for eachprimer pair), 400 mM dNTPs, 1×PCR buffer containing MgCl2, and 0.2 U TaqDNA polymerase. PCR was performed on the ABI GeneAmp 9700 PCR Systemunder the following conditions: 5 min at 94° C.; 30 cycles of 30 sec at94° C., 1 min at 60° C., 1 min at 72° C.; and 5 min at 72° C. The PCRproducts were separated by capillary electrophoresis on an ABI 3130XLDNA Analyzer. Chromatograms were analyzed with Peak Scanner 1.0 andGeneMapper 3.7 software (Applied Biosystems) for allelic alterations.The area of the peaks produced by the DNA PCR products was quantifiedfor each allele. The ratio of the allelic areas was calculated for eachtumor and paired normal DNA sample. When the qLOH (allelic ratio for thetumor peaks divided by the allelic ratio of paired normal sample) was≤0.5 or ≥2.0 for c-CBL and at least one other 11q marker in at least twoseparate experiments, the sample was considered as having an allelicimbalance and interpreted as LOH. Samples were evaluated in at least twoseparate experiments and samples showing prospective LOH at c-CBLrepeated a third time which included a new control marker at the BAXlocus on chromosome 19 to verify integrity of sample DNA.

Transfections of c-CBL Constructs

The A549 cell line was transfected using the Fugene HD (Roche, Nutley,N.J.) reagent according to the manufacturer's instructions. Eight mg ofplasmid DNA, containing either no insert (empty vector), wild-typec-CBL, S80N/H94Y c-CBL, Q249E c-CBL or W802*CBL was used fortransfection in a 6-well culture plate. Cells were harvested 48 h aftertransfection and analyzed for expression.

c-CBL Knockdown

c-CBL knockdown was performed using lentiviral transduction usingMISSION lentiviral transduction particles (Sigma-Aldrich, St. Louis,Mo.) as per manufacturer's instructions. Briefly, 16105 H358 cells/wellwere seeded in 6-well plates and infected the following day with c-CBLlentiviral shRNA constructs. To generate stable c-CBL knockdown celllines, cells were selected for 2 days with 1 mg/ml puromycin. c-CBLlevels were determined using whole cell lysates by immunoblotting withanti-CBL antibody (Santa Cruz Biotechnologies, Santa Cruz, Calif.).

Cell Viability Assay

Cells were transfected as described above in the transfection assay.Forty-eight hours after transfection, viability of cells was assessedusing Trypan Blue exclusion.

Wound Healing Assay

A549 cells were seeded in 6-well plates and cultured for 48 h until 100%confluent. The medium was then changed and the cells were transfected asdescribed in the transfection assay. Twelve hours after transfection, astraight scratch was made across the cell layer using a 1 ml pipettetip. The cells were then gently washed with 16 PBS to remove cellulardebris and the media was replaced. Photographs were taken of the woundregion every 12 h until 48 h.

Western Blot Analysis

Forty eight hours after transfection, cells were collected and washedtwice in 1×PBS, then lysed in ice-cold lysis buffer (0.5M Tris-HCl withpH 7.4, 1.5 M NaCl, 2.5% deoxycholic acid, 10 mM EDTA, 10% NP-40, 0.5 mMDTT, 1 mM phenylmethylsulfonyl fluoride, 5 mg/mL leupeptin, and 10 mg/mLaprotinin) for 5 minutes. The lysate was centrifuged at 13,000 rpm for20 minutes at 4° C., and protein content of the supernatant wasmeasured. Total cell lysates (50 mg/well) were separated by SDSPAGEelectrophoresis and the gels transferred onto nitrocellulose membranes(Whatman, Piscataway, N.J.). Membranes were blocked with 5% non-fat drymilk in phosphate-buffered saline containing Tween-20 (PBST) (1×PBS,0.1% Tween-20) for 1 h at room temperature and incubated with theappropriate primary antibody at 4° C. overnight. Membranes then werewashed three times with PBST and probed with appropriate horseradishperoxidase (HRP)-conjugated secondary antibody for 1 h at roomtemperature. The membranes were again washed three times in PBST andbands were visualized using Western blot chemiluminescence reagent(BioRad, Valencia, Calif.) on a Chemidoc Gel documentation system(BioRad, Valencia, Calif.). Antibodies were obtained from Santa CruzBiotechnologies and used at the following dilutions: c-CBL, 1:5000;c-MET, 1:5000; EGFR, 1:5000; ubiquitin, 1:1000; HA, 1:5000 and b-actin,1:10,000.

Flow Cytometry

Cell cycle analysis was carried out by flow cytometry. Approximately26106 cells were grown in media containing 10% FBS. Cells were harvestedby trypsin/EDTA treatment, washed with 1×PBS three times and fixed withice-cold 70% ethanol for 2 h. Cells were washed again with cold PBS andstained with a solution containing 25 mg/mL propidium iodide, 200 mg/mLRNase A, and 0.1% Triton X-100 for 30 minutes in the dark. Cell cycleanalysis was performed using a Guava PCA-96 flow cytometer (GuavaTechnologies, Millipore, Billerica, Mass.).

Ubiquitin Ligase Activity

293T cells were maintained in culture in DMEM supplemented with 10% FBSand 1% penicillin (100 units/mL) and streptomycin (100 mg/mL) weretransfected with 0.2 mg EGFR-pcDNA3 and 2 mg HA-tagged c-CBL constructsas indicated using calcium phosphate according to manufacturer'sprotocol (Profection, Promega, Madison, Wis.). Twenty-four hourspost-transfection, cells were starved overnight in DMEM supplementedwith 0.5% FBS, and then treated with or without EGF (100 ng/ml) for 15min. The cells were collected and washed two times in ice-cold PBScontaining 0.2 mM sodium orthovanadate then lysed in icecold lysisbuffer (10 mM Tris HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X100,10% Glycerol, 2 mM sodium orthovanadate and protease inhibitors).Lysates were cleared of debris by centrifugation at 16,000 g for 10 minat 4° C. EGFR immunoprecipitations were performed on 200 mg of clearedlysate using 250 ng of rabbit-anti-EGFR and Protein A/G Plus Sepharoseovernight at 4 uC. Precipitations were washed 5 times in lysis bufferbefore boiling in Laemmli buffer. Elutions were immunoblotted withanti-ubiquitin and EGFR. Twenty micrograms of cleared lysate wereimmunoblotted for each of the c-CBL constructs using anti-HA.

Statistical Analysis

Mutation rates between different groups were compared using Fisher'sexact test. For continuous variables, group comparisons were performedusing analysis of variance (ANOVA) followed by Sidak's adjustment formultiple comparisons. Experiments involving repeated measurements overtime were analyzed using repeated measures ANOVA with theGreenhouse-Geisser adjustment to the degrees of freedom. Analyses wereconducted using STATA (v10.1) software (Stata Corporation, CollegeStation, Tex.).

c-CBL Gene Mutations in Lung Cancer

To investigate the role of c-CBL in lung cancer, its genomic DNA intumor and paired normal samples drawn from multiple ethnicities wasanalyzed. The lung tumor samples represented Caucasians (n=50),African-Americans (n=29), and Taiwanese (n=40) lung cancer patients.Twelve pairs of primers were designed to sequence the coding region ofc-CBL gene that spans exons 2 to 16. Eight unique somatic mutations inc-CBL exons were identified among 8 different patients. A variationL620F, a known SNP (rs2227988) in exon 11 was also detected.Importantly, the eight novel nonsynonymous mutations were confirmed bysequencing both strands of c-CBL genomic DNA obtained from lung tumorsamples (Table 1). Moreover, none of the 8 mutations were detected inthe corresponding normal tissue, indicating that these were somaticmutations. Four synonymous single nucleotide variations (SNVs) were alsoidentified.

TABLE 1 c-CBL mutation analysis in 119 lung cancer patient tumortissues. Numbers of Sample (Frequency) Location Mutation DomainCaucasian (5.0)* African-American (20)* Taiwanese (40)* Exon 2 26354 G >AG (S80N)^(#) TKB 1 (2%) 0 0 26395 C > CT (M94Y)^(#) TKB 1 (2%) 0 0 Exon4 67885 C > CG (Q249E) TKB 0 0 1 (2.5%) Exon 8 72104 G > AG (V391I) RING1 (2%) 1 (3.5%) 0 Exon 9 72515_72517 del ATG Pro-rich 1 (2%) 0 0 Exon 1178346 C > CT (L620F){circumflex over ( )} Pro-rich 0 0 2 (5%) Exon 1592375 G > AG (W802*) C-terminal 0 1 (3.5%) 0 Exon 16 93412 G > AG(R830K) C-terminal 0 1 (3.5%) 0 93466 G > AG (A848T) C-terminal 0 1(3.5%) 0 *( ) Indicates number of samples. ^(#)S80N and H24Y mutationswere found in the same patient. {circumflex over ( )}Known SNP.

Three of the 8 non-synonymous mutations were located in the TKB(tyrosine kinase binding) domain (S80N, H94Y, and Q249E), one in theRING finger domain (V391I), one in the proline-rich region (72515_72517del ATG), and three in the Cterminal region (W802*, R830K, and A848T) ofthe c-CBL protein (FIG. 5A). In FIG. 5B, we show model chromatograms ofrepresentative samples.

11q LOH of c-CBL Gene

Paired lung tumor and normal lung tissue samples from Taiwanese patients(n=37) were investigated for LOH. Eight (21.6%) showed LOH at the c-CBLlocus on chromosome 11 while 29 samples (78.4%) revealed normal alleliccontribution at the microsatellite markers (FIGS. 5C and D).

c-CBL Mutations in Different Ethnic Groups

The c-CBL double mutant S80N/H94Y was found in the same patient, and theoverall mutation rate for c-CBL in lung tumors was 6.7% (8/119). Thefrequency of c-CBL mutation was highest in large cell carcinoma (14.7%;5 of 34 patients), followed by squamous carcinoma (6.3%; 2 of 32patients), and the least was observed in adenocarcinoma (AD) (1.8%; 1 of53 patients), although these rates were not statistically significant(p=0.292). Mutation rates were 6.0% among Caucasians (0 of 20 in AD; 0of 10 in SQ; and 3 of 20 in LC), 13.8% in African-Americans (1 of 10 inAD; 1 of 10 in SQ; and 2 of 9 in LC), and 2.5% (0 of 23 in AD; 1 of 12in SQ; and 0 of 5 in LC) in the Taiwanese population. Additionally, twoTaiwanese patients with lung cancer (one squamous and oneadenocarcinoma) had the known SNP L620F.

Mutations in MET and EGFR can be Co-Associated with c-CBL Alterations

Since East Asians with lung cancer have a higher frequency of EGFR andMET mutations in lung tumors, mutations in EGFR and MET in the sameTaiwanese cohort samples were also determined. These results werecompared with the observed c-CBL alterations (LOH and/or mutations). Inthe 37 samples tested, no overlap between c-CBL mutations and c-CBL LOHwas found (FIG. 6). Of the three c-CBL mutants (including the knownL620F SNP, rs2227988), one of the samples had a MET mutation (N375S),and the other had an EGFR mutation (L858R). Among the 8 samples that hada LOH at the c-CBL locus, 5 had an additional mutation in MET (N375S),and 2 had an EGFR exon 19 deletion. Twenty-six samples had neither c-CBLmutation nor c-CBL LOH (3 patients had a c-CBL mutation but no c-CBLLOH). Among these 26 samples 9 had a MET mutation (8 N375S, 1 L211W), 13had an EGFR mutation (7 exon 9 deletion, 6 L858R), and 4 had no otherMET or EGFR mutation. Thus, the rate of MET or EGFR mutations amongpatients with LOH at the c-CBL locus (7 of 8) was similar to that seenin patients without c-CBL mutation or LOH (22 of 26 patients) (p=0.99).These 4 patients with no identifiable mutation in c-CBL, MET or EGFRrepresented 10.8% of the 37 patients analyzed in the Taiwanese patientcohort. Conversely, 89.2% Taiwanese lung cancer patients have anidentifiable mutation in either c-CBL, MET or EGFR or a combination ofthe three genes (FIG. 6). Additionally, p53 and KRAS mutations weredetermined in these Taiwanese cohorts. Two p53 and 1 KRAS mutation weredetected. The single KRAS mutation overlapped with one p53 mutation.This patient also had the EGFR exon 19 deletion but had no c-CBLmutation. The other p53 mutation sample had a c-CBL LOH with concurrentMET N375S mutation. Thus, in the Taiwanese samples analyzed, p53/KRASmutations and c-CBL mutations were mutually exclusive.

Cellular Functions of c-CBL Alterations in the Context of LungTumorigenesis

E3 activity is intact in the mutant c-CBL proteins. To investigatewhether the different c-CBL mutations affect the E3 activity, EGFR waschosen as a model substrate for c-CBL E3 function. All of the c-CBLmutants tested enhanced ubiquitination of the activated EGFR similar tothe wild-type c-CBL protein. The catalytic activity of the c-CBL mutantswas not impaired when EGFR was the substrate.

Effect on Lung Cancer Cell Viability.

The effect of a representative c-CBL mutant from each of the threeethnic backgrounds on lung cancer cell viability in cell lines wasdetermined. S80N/H94Y double mutation, Q249E, and W802* were identifiedin lung tumor samples obtained from a Caucasian, a Taiwanese and anAfrican-American, respectively. As described above, the c-CBL wild-type(WT) and the above three mutants were expressed after cloning them intopAlterMax vector in A549 cells. These cells express relatively low basallevels of endogenous c-CBL. Transfection efficiency was comparablebetween different groups, and the number of cells transfected with c-CBLwild-type construct was about 70% compared to control cells that weretransfected with the empty vector. Cells transfected with S80N/H94Y,Q249E and W802*c-CBL mutant constructs resulted in increased number ofviable cells that was 132.3%, 120.8% and 147.9% higher respectively,relative to the empty vector control-transfected cells and significantlydifferent from the wild-type construct (p=0.022, p=0.049, and p=0.008,respectively) (FIG. 7B). Relative levels of c-CBL protein in whole celllysates prepared from samples obtained from a parallel experiment weredetermined. The c-CBL protein levels in samples representinguntransfected and empty vector transfected cells were comparable, andthose representing the c-CBL WT and the three c-CBL mutants werecomparable (FIG. 7C).

Effect on Cell Cycle.

To investigate if the increases in cell viability in different c-CBLmutants are due to increased cellular proliferation, a cell cycleanalysis was performed. A549 cells were transfected with the c-CBL WT orthe three different mutants: S80N/H94Y, Q249E and W802*. The emptyvector transfectant was used as a control. Forty-eight hours aftertransfection, cell cycle analysis was performed as described herein.There was no significant change in the subG1, G1 or the S phase of thecell cycle among the different mutants compared to the WT construct(p=0.64, p=0.40, and p=0.28, respectively). The G2/M phase of the cellcycle showed an increase in cell numbers for the three mutants,S80N/H94Y, Q249E and W802*, when compared to the WT but again thedifference was not statistically significant (p=0.25) (FIG. 7D).

Effect on Cell Motility.

To investigate the effect of the expression of the above three c-CBLmutants on cell migration, a wound healing assay was carried out asdescribed herein. The closing of the scratch or the wound was monitoredat 0, 12, 24, 36, and 48 h. (FIG. 8A). In all the samples thatrepresented cells transfected with mutants, the wound gap was muchsmaller than that seen in the sample that represented cells transfectedwith c-CBL WT (p<0.001). The rate of wound closure was also determinedfor all the five groups. At 48 h, wild-type c-CBL transfectants showed61.1% open wound, while the S80N/H94Y, Q249E and W802* mutants showed18.7%, 23.9% and 34.3% open wound, respectively (p<0.001) (FIG. 8B).

c-CBL Knockdown Increases Cell Viability.

The effect of c-CBL knockdown in lung cancer cells was tested. Comparedto A549, H358 lung cancer cells express relatively high levels ofendogenous c-CBL. c-CBL expression was knocked down using lentiviralconstruct that expressed c-CBL specific shRNA, and this expression wascompared to the expression in cells that were transduced with scrambledshRNA. Several clones that revealed varying degrees of c-CBL knockdownshowing different levels of c-CBL lentiviral shRNA knockdown efficiencywere identified (FIG. 9A). Of all the clones tested, Clone 27 was chosenfor further experiments. Equal amounts of cells were seeded in a 6-wellplate, and the cell proliferation was measured at various times and theresults are depicted in FIG. 9B. The number of cells increased in a timedependent fashion from 100 to 190% relative to scrambled shRNA ascontrol in a span of 48 h (p=0.0002) (FIG. 9B). The cell cycle phases inH358 cells that were knocked down with c-CBL shRNA were analyzed andcompared with the scrambled shRNA. There were no discernable differencesbetween these two constructs in the different phases of the cell cycle.

The results demonstrated that c-CBL was somatically mutated (or had LOH)in lung cancer cells. Accordingly, it is contemplated that c-CBL cansignificantly contribute to enhanced cell viability and motility. Theresults also demonstrated that there was also a high prevalence of LOHwith respect to c-CBL in lung tumors that harbored MET or EGFRmutations.

The results demonstrated the occurrence of c-CBL mutations in lungcancer patients, especially with different ancestral variations.Mutations in c-CBL have been recently reported in juvenilemyelomonocytic leukemia and myeloid malignancies. In the AML study, themutation R420Q located in the junction of the RING finger and the linkerregion inhibited FMS-like tyrosine kinase 3 (FLT3) internalization andubiquitination (Sargin et al., 2007), thus contributing to thegain-in-function for the RTK. In addition, mutations such as H398Y,C384R, and L380P have been mapped to the RING finger domain and thelinker region of c-CBL that is required for its E3 activity. (CaligiuriM A, et al., Blood 110: 1022-1024 (2007); Grand F H, et al. Blood113:6182-6192 (2009); Dunbar A J, et al., Cancer Res 68: 10349-10357(2008); Sanada M, et al. (2009) Nature 460:904-908; and Reindl C, et al.(2009) Clin Cancer Res 15: 2238-2247). Additionally, homozygousmutations in the RING finger domain of the c-CBL gene were described asa result of acquired Uniparental Disomy (UPD). The results indicated LOHat 11q23 locus, and these are mutually exclusive from missense mutationsof c-CBL. The somatic mutations were all heterozygous. The mutations inAML led to abrogation of the E3 activity, leading to prolonged RTKactivation. In addition mutants located on the linker region surroundingthe RING finger domain exhibited enhanced AKT signaling in response tocytokine stimulation. In addition, in NH3T3 cells, it has been shownthat neither mutations in the RING finger nor the linker region causetransformation; however, while certain mutations perturb theubiquitination, others affect receptor recycling and prolong kinaseactivity. (Thien C B and Langdon W Y (2001) Nat Rev Mol Cell Biol 2:294-307).

The results demonstrated that some c-CBL mutations were mapped not onlyto the RING finger domain, but also to the TKB domain, proline-richdomain and the C-terminal region, but none mapped to the linker regionas reported in the AML studies described above (Caligiuri et al., 2007;Grand et al., 2009; Dunbar et al., 2008; Sanada et al., 2009; Reindl C,et al. (2009) Clin Cancer Res 15: 2238-2247). In addition, eight mutantsthat we detected were found in different ethnic backgrounds. Forexample, S80N/H94Y, Q249E, and W802* were detected in Caucasians,Taiwanese and African-Americans, respectively. The results demonstratenot only the difference between lung cancer and other cancers, but alsogenetic polymorphism among different races in the same cancer. There isa large disparity between African-American and other ethnic populationswith lung cancer. It has previously been shown that there is a lowfrequency of EGFR and MET mutation in African-Americans as compared withTaiwanese and Caucasians. In the results described herein, threemutations that are unique to the African-American ethnicity were found.

c-CBL plays an important role in down regulating RTK mediated signalingthrough K63 poly-ubiquitination and subsequent downregulation of RTKsfollowed by lysosomal degradation. Mono-ubiquitination or ubiquitinatedwith K63-linked chains of substrates by c-CBL may lead to enhancement ofbiological and biochemical functions. Ubiquitin and ubiquitin-likeproteins in protein regulation. The analyzed mutations indicated thatthe E3 activity of c-CBL on EGFR remained intact and that the EGFRlevels in the various mutants remained the same. It is contemplated thatmultiple kinases, both RTKs and non-RTKs, could be acted upon by c-CBL,including ERBs, PDGFR, FMS, MET, c-Kit, VEGFR, FLT-1, RON, FGFR, IR, aswell as SYK, FYN, LCK, FGR, LYN and c-ABL.

It has been previously shown that activating c-CBL mutationdownregulates EGFR signaling and decreases cellular proliferation andmigration in breast cancer cell lines. Although the role of c-CBL in thenegative regulation of RTKs (and as a potential tumor suppressor) iswell substantiated, studies in cancer cells have revealed both tumorsuppressor and tumor promoting activities, depending on the type ofc-CBL mutation and the number of alleles at the c-CBL locus. Consistentwith these previous studies, the results related to the three c-CBLmutants described herein demonstrate that the mutants have both tumorgrowth and metastasis promoting properties. Although these mutants areoutside of the RING finger or the linker region of c-CBL, it iscontemplated that their downstream effects can be significant so as tocause increased proliferation and migration. The results also indicatedthat LOH for c-CBL was found in a significant number of samples thatharbored MET or EGFR mutations. It is contemplated that about 7% of lungtumor samples can be likely to have c-CBL mutations and an additional22% can be likely to harbor c-CBL-related LOH, making c-CBL a highlymutated molecule in lung cancer.

Previous studies have shown that East Asians with lung cancers haverelatively high frequencies of gain-of-function of mutations in RTKssuch as EGFR and MET. In a cohort of Japanese patients, an activatingMET mutation was identified in the splice region that deletes thejuxtamembrane domain that is involved E3 activity of c-CBL. The samestudy also found that activation of MET is mutually exclusive of EGFR,KRAS and HER2 gene mutations. During the experiments described herein,significant numbers of such mutations in lung tumor samples obtainedfrom African-Americans (n=29) and Caucasian (n=50) patients were notdetected. In the experiments described herein, one MET mutation wasidentified in each of the groups whereas 1 and 3 EGFR mutations wereidentified in the African-American and Caucasian cohorts, respectively.EGFR mutations have previously been identified as one of the keymutations affecting lung adenocarcinoma patients in a comprehensivestudy of 188 patients. The experiments described herein encompasseddifferent histologies of NSCLC. However, the published series did notfind any mutations in c-CBL or MET; in contrast, the experimentsdescribed herein encompassed different subtypes of NSCLC. It hasrecently been shown that MET mutations in lung cancer are in majoritygermline. To better understand the ethnic differences in the lungoncogenome, PAX transcription factors such as PAX5 and PAX8 that arehighly expressed in lung cancers were examined; however, the resultsindicated that there was no preferential expression or mutations of theabove genes in lung tumor samples of African-Americans. The results diddemonstrate a relatively high frequency of c-CBL mutations in lungcancers, especially in the large cell type among Caucasians andparticularly among African-Americans.

Example 3

Adenocarcinoma and undifferentiated NSCLC were stained with c-CBL andMET antibodies on whole tissue sections. The results demonstratedstronger MET expression throughout the tissue sections with weaker c-CBLstaining in localized areas of the section (n=29, with 11adenocarcinomas, 11 squamous cell carcinomas, and 7 large cellcarcinomas). c-CBL staining was intense in the lymphocytes (L) and weakin the tumor (T) whereas the staining pattern was the reverse for MET.FIG. 10A shows representative c-CBL and MET staining in two differentpatient samples, one adenocarcinoma sample and one NSCLC sample. Thestaining intensity was analyzed in these 29 samples among threedifferent histologies, and the results show relatively intense METstaining with relatively low to moderate c-CBL staining. The results aresummarized in FIG. 10B. The relative intensity of expression of c-CBLwas higher in large cell carcinoma as compared to other histologies.Also, c-CBL mutations were greater in large cell carcinomas.

The results from the archival database at www.proteinatlas.org weresupportive of the results described herein, which found negative or lowstaining for c-CBL in NSCLC. The staining of c-CBL was relatively lowcompared to CBL-b and CBL-c staining (FIG. 11). Specifically, METstaining was more intense in squamous cell carcinoma (SQ, n=11) andadenocarcinoma (AD, n=11) compared to c-CBL, which was low to moderatein most samples except large cell (LC, n=7).

Example 4

Nude mice were injected intravenously (tail vein) with 10 million A549NSCLC cells stably transfected with luciferase (luc) construct. Thehoming of these cancer cells to lungs, lymph nodes and the spleen wereimaged at regular intervals (once weekly) by whole body imaging usingiBOX imaging system. The mice were also injected subcutaneously tomeasure tumor growth rates in different cell lines as well as lineshaving different RTK mutations. Animals were examined daily for sixweeks for weight loss and other signs of morbidity and were terminatedif found to be distressed. All animals were terminated at 6 weeks in ahumane way. At weekly intervals, the tumors were scanned by whole bodyimagers using the iBOX imaging system. After subtracting backgroundfluorescence, signals in the images were calculated using Igor Prosoftware, and the tumor size was determined. A representative exampleimage is shown in FIG. 12.

Example 5

The effects of silencing wt c-CBL in combination with a variety of drugsthat inhibit EGFR (erlotinib [Tarceva]), PI-3K (NVP, Wortmanin), MET(SU11274) on the growth of H358 NSCLC cells that endogenously expressc-CBL-Wt were investigated. The H358 cells, which expressed high levelsof c-CBL, were knocked down using a lentiviral construct (sh 27,directed against c-CBL-Wt in H358 clone 27 cells (black), or the sh SCRscrambled RNA control (white)). Cells were treated with RTK and P1-3Kinhibitors, cisplatin (CDDP). Live cells were assessed 72 hours afterdrug treatment using an MTT assay. The results are summarized in FIG.13. As shown in FIG. 13, silencing c-CBL-Wt did not have a discernibleeffect on the percentage of live cells (media control); however,silencing c-CBL-Wt significantly reduced the ability of SU11274 (alsoreferred to as SU) to kill NSCLC cells. A comparison of the c-CBL-Wtexpressing cells (white) and the c-CBL-Wt knockdown cells (black)treated with SU11274 either alone or in combination with othertreatments, showed that c-CBL-Wt knockdown cells were less susceptibleto treatment with a c-MET inhibitor.

Example 6 Tissue Samples

Tumor and paired adjacent normal tissues were obtained from patientsthat received treatment.

Tissue Microarray and Immunohistochemistry

Tissue blocks of patients with HNSCC that were treated at the Universityof Chicago Medical Center (diagnosed between 1992 and 2005) wereselected for the study. Tumor microarray (TMA) was built using theATA-27 Arrayer from Beecher Instruments as previously described (Ma etal., Genes Chromosomes Cancer 2008). In brief, tissue cores (1.5-mmpunch) from biopsied tumor and adjacent normal tissues were preciselyorganized into a grid and embedded in paraffin. Each specimen wasincluded in duplicate within the array. All slides were reviewed andscored by two independent pathologists. Differences inimmunohistochemical scores were resolved by consensus. Each sample wasscored using a 0 to 3+ scale, with 0 denoting no staining/no proteinexpression and 3+ denoting strong positive staining/high proteinexpression.

Immunoblotting

Cells were harvested and washed in 1×PBS, then lysed in ice-cold M-PERlysis buffer plus HALT protease and HALT phosphatase inhibitors (Pierce)for 10 minutes on ice. The lysates were centrifuged at 13,000 rpm for 10minutes at 4° C., and protein content of the supernatant was determined.Total cell lysates (50 μg/well) were separated by SDS-PAGEelectrophoresis and the gels transferred onto immobilon-P membranes(Whatman, Piscataway, N.J.). Membranes were blocked with 5% BSA inTris-buffered saline containing Tween-20 (TBST) (1×TBS, 0.1% Tween-20)for 1 h at room temperature and incubated with the appropriate primaryantibody at 4° C. overnight. Membranes were then washed three times withTBST and probed with the appropriate horseradish peroxidase(HRP)-conjugated secondary antibody (1:10,000) for 1 h at roomtemperature. The membranes were again washed three times in TBST andbands were visualized using Western blot chemiluminescence reagent(BioRad, Valencia, Calif.) on a Chemidoc Gel documentation system(BioRad). The following antibodies were used: c-CBL (Abcam), 1:500; MET(Invitrogen), 1:500; and β-actin (Sigma), 1:10,000.

c-CBL Gene Mutational Analysis

Genomic DNA was isolated from formalin-fixed, paraffin embedded (FFPE)patient tissues using the QIAamp DNA Minikit (Qiagen) according to themanufacturer's instructions. Exons 2 to 16 of c-CBL were amplified andsequenced as previously reported (Tan et al., PLoS One 2010). The PCRconditions were as follows: 1 cycle—95° C. for 5 min; 30 cycles—95° C.for 30 s, 58° C. for 30 s, 72° C. for 1 min; 1 cycle—72° C. for 5 min.Sequencing was performed on the forward coding strand with confirmationof c-CBL alterations performed by sequencing the reverse strand.Mutational analysis was performed using Mutation Surveyor v2.61(Softgenetics, State College, Pa.).

Loss of Heterozygosity (LOH) Analysis

c-CBL LOH analysis was conducted as previously described (Tan et al.,PLoS One 2010). Briefly, five microsatellites on chromosome 11 (3 withinthe c-CBL gene on 11q and 2 control markers on 11p) were selected foranalysis (FIG. 5D). Genomic DNA was extracted from FFPE tumor samplesand paired normal tissue. Marker D11S929 served as an internal controlto check for consistency in PCRs and of peaks from capillaryelectrophoresis. PCRs were carried out in a volume of 10 μL thatcontained 1 μL genomic DNA (20-50 ng), 0.5 μM of each primer (1.0 μMtotal for each primer pair), 400 μM dNTPs, 1×PCR buffer containingMgCl2, and 0.2 U Taq DNA polymerase. The PCR conditions were: 5 min at95° C.; 30 cycles of 30 sec at 95° C., 1 min at 60° C., 1 min at 72° C.;and 5 min at 72° C. Peak Scanner 1.0 (Applied Biosystems) was used toanalyze the chromatograms. The ratio of the allelic areas was calculatedfor each tumor and paired normal DNA sample. When the qLOH (allelicratio for the tumor peaks divided by the allelic ratio of paired normalsample) was ≤0.5 or ≥2.0 for c-CBL and at least one other 11q marker inat least two separate experiments, the sample was considered as havingan allelic imbalance and interpreted as LOH.

Example 7

Cell Viability of Various c-CBL Mutants

The effect of representative c-CBL mutants (c-CBLMts), S80N/H94Y (SH)double mutation, Q249E (Q), V391I (V) and W802* (W*), on lung cancercell viability in A549 and H226 nonsmall cell lung cancer cell lines wasdetermined. Cell viability was measured by trypan blue exclusion andcompared to empty vector (EV) control. c-CBL wild type (WT) and mutantsS80N/H94Y (SH), Q249E (Q), V391I (V), and W802* (W*) showed 69%, 104.8%,100.6%, 89.7%, and 109.1% cell viability respectively in A549 and 50%,106.9%, 84.5%, 70.7%, and 60.3% respectively in H226 cells after 48 htransiently transfection.

c-CBLMts Exhibit Decreased c-Met Ubiquitination in NSCLC

A549 cells were transiently transfected with empty vector (EV) or c-CBLwild type (WT) and mutants' constructs (SH: S80N/H94Y, Q: Q249E, V:V391I, W*: W802*). At 48 h, whole cell lysates were immunoprecipitated(IP) with anti-c-Met Ab and immunoblotted (IB) with anti-ubiquitin Ab.IB with anti-HA Ab for transfection efficiency and β-actin for loadingcontrol of the IP. The results showed the ubiquitination of c-Met weredecreased in A549 cells that transiently expressed c-CBL mutantsrelative to wild type c-CBL cells.

Cell Survival of c-CBLMts after Treatment with c-Met and EGFR Inhibitors

A549 and H226 cells were transiently transfected with empty vector (EV)or c-CBL wild type (WT) and mutants' constructs (SH: S80N/H94Y, Q:Q249E, V: V391I, W*: W802*). At 24 h, cells were recollected and seeded5×104 cells/well in 24 well culture plate. Another 24 h, cells weretreated with c-Met inhibitor SU11274 and EGFR inhibitor Tarceva indifferent concentrations. Cell survival was detected by cell countingafter 48 h treatment. The results in A549 cells showed c-CBL mutants SH,Q, V, W* had more sensitivity to the c-Met inhibitor SU11274 than didA549 and H226 cells transfected with the wild type c-CBL. However, thereis no difference with EGFR inhibitor Tarceva treatment. Transientlyexpressed c-CBL mutants showed a decrease in the ubiquitination of c-Metrelative to wild type c-CBL cells (FIG. 21) A549 and H226 cells aregenerally accepted predictive cell models for carcinoma behavior, forexample, NSCLC and head and neck carcinoma. The susceptibility of c-CBLmutant A549 and H226 cells to treatment with a c-MET inhibitor, forexample, SU11274, is predictive of the susceptibility of a subject, suchas a patient, diagnosed with cancer or having cancer to treatment with ac-MET inhibitor, for example, SU11274.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific embodiments described specifically herein. Such equivalents areintended to be encompassed in the scope of the following claims.

1. A method of identifying a subject that is susceptible to treatmentwith a c-Met inhibitor comprising determining whether a sample from thesubject comprises a mutation in a nucleic acid sequence encoding humanc-CBL, wherein the mutation results in an amino acid change at positionS80N, H94Y, Q249E, V391I, 72515_72517delATG, W802*, R830K, A848T, L620F,P170L, S171S, L281F, L254S, or P782L.
 2. The method of claim 1, whereinthe presence of a mutation in a nucleic acid sequence encoding humanc-CBL identifies a subject that is susceptible to treatment with a c-Metinhibitor.
 3. The method of claim 1, wherein the absence of a mutationin a nucleic acid sequence encoding human c-CBL identifies a subjectthat is not susceptible to treatment with a c-Met inhibitor.
 4. Themethod of claim 1, wherein the subject has been diagnosed with cancer.5. The method of claim 1, wherein the subject has cancer.
 6. The methodof claim 1, wherein the sample is a cancer sample.
 7. A method ofidentifying a subject that is susceptible to treatment with a c-Metinhibitor comprising determining whether a sample from the subjectcomprises a mutation in a nucleic acid sequence encoding human c-CBL,wherein the mutation is located in the TKB domain, RING finger domain,proline-rich region, C-terminal region, or other domain linkage regionsof c-CBL.
 8. The method of claim 7, wherein the presence of a mutationin a nucleic acid sequence encoding human c-CBL identifies a subjectthat is susceptible to treatment with a c-Met inhibitor.
 9. The methodof claim 7, wherein the absence of a mutation in a nucleic acid sequenceencoding human c-CBL identifies a subject that is not susceptible totreatment with a c-Met inhibitor.
 10. The method of claim 7, wherein themutation results in an amino acid change at position S80N, H94Y, Q249E,V391I, 72515_72517delATG, W802*, R830K, A848T, L620F, P170L, S171S,L281F, L254S, or P782L. 11-16. (canceled)
 17. A method of determiningresponsiveness of a cancer in a subject to treatment with a c-Metinhibitor, said method comprising determining whether a cancer samplefrom a subject comprises a mutation in a nucleic acid sequence encodinghuman c-CBL, wherein the mutation is located in the TKB domain, RINGfinger domain, proline-rich region, C-terminal region, or other domainlinkage regions of c-CBL, wherein the presence of the mutated nucleicacid sequence is indicative that the cancer is responsive to treatmentwith the c-Met inhibitor. 18-28. (canceled)